Binding assay employing IGS4, a human G-protein coupled neuromedin receptor

ABSTRACT

The present invention relates to novel identified polynucleotides, polypeptides encoded by them and to the use of such- polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the G-protein coupled receptor family, referred to as G-family. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides, to a vector containing said polynucleotides, a host cell containing such vector and transgenic animals where the IGS 4 -gene is either overexpressed, misexpressed, underexpressed or suppressed (knock-out animals). The invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor family IGS 4 . The invention also relates to the identification of the cognate ligand of the IGS 4  polypeptides of the invention. High affinity binding to said IGS 4  polypeptides is found for the neuropeptides known as neuromedin U.

This is a division of application Ser. No. 10/088,744, which has a 35U.S.C. § 371 filing date of Mar. 22, 2002, now U.S. Pat. No. 6,998,255is a 35 U.S.C. § 371 filing of PCT/EP00/09584, filed Sep. 25, 2000 whichclaims priority to EP application No. 99203140.1, filed Sep. 24, 1999,Netherlands application No. 1013140, filed Sep. 24, 1999, EP applicationNo. 00202683.9, filed Jul. 28, 2000, and U.S. application No.60/222,047, filed Jul. 31, 2000, all of which are incorporated herein byreference.

The present invention relates to novel identified polynucleotides,polypeptides encoded by them and to the use of such polynucleotides andpolypeptides, and to their production. More particularly, thepolynucleotides and polypeptides of the present invention relate to aG-protein coupled receptor (GPCR), hereinafter referred to as IGS4. IGS4exists in two polymorphic forms, hereinafter referred to as IGS4A andIGS4B. The invention also relates to inhibiting or activating the actionof such polynucleotides and polypeptides, to a vector containing saidpolynucleotides, a host cell containing such vector and transgenicanimals where the IGS4-gene is either overexpressed, misexpressed,underexpressed and/or suppressed (knock-out animals). The inventionfurther relates to a method for screening compounds capable to act as anagonist or an antagonist of said G-protein coupled receptor IGS4, and tothe cognate ligand of IGS4.

BACKGROUND OF THE INVENTION

It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers; e.g., cAMP(Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins arereferred to as proteins participating in pathways with G-proteins. Someexamples of these proteins include the GPC receptors; such as those foradrenergic agents and dopamine (Kobilka, B. K., et al., Proc. Natl.Acad. Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al., Science, 1987,238:650-656; Bunzow, J. R., et al., Nature, 1988, 336:783-787),G-proteins themselves, effector proteins, e.g., phospholipase C,adenylate cyclase, and phosphodiesterase, and actuator proteins, e.g.,protein kinase A and protein kinase C (Simon, M. I., et al., Science,1991, 252:802-8).

For example, in one form of signal transduction, upon hormone binding toa GPCR the receptor interacts with the heterotrimeric G-protein andinduces the dissociation of GDP from the guanine nucleotide-bindingsite. At normal cellular concentrations of guanine nucleotides, GTPfills the site immediately. Binding of GTP to the α subunit of theG-protein causes the dissociation of the G-protein from the receptor andthe dissociation of the G-protein into α and βγ subunits. TheGTP-carrying form then binds to activated adenylate cyclase. Hydrolysisof GTP to GDP, catalyzed by the G-protein itself (α subunit possesses anintrinsic GTPase activity), returns the G-protein to its basal, inactiveform. The GTPase activity of the α subunit is, in essence, an internalclock that controls an on/off switch. The GDP bound form of the αsubunit has high affinity for βγ and subsequent reassociation of αGDPwith βγ returns the system to the basal state. Thus the G-protein servesa dual role, as an intermediate that relays the signal from receptor toeffector (in this example adenylate cyclase), and as a clock thatcontrols the duration of the signal.

The membrane bound superfamily of G-protein coupled receptors has beencharacterized as having seven putative transmembrane domains. Thedomains are believed to represent transmembrane α-helices connected byextracellular or cytoplasmic loops. G-protein coupled receptors includea wide range of biologically active receptors, such as hormone, viral,growth factor and neuroreceptors.

The G-protein coupled receptor family includes dopamine receptors whichbind to neuroleptic drugs used for treating CNS disorders. Otherexamples of members of this family include, but are not limited tocalcitonin, adrenergic, neuropeptideY, somastotatin, neurotensin,neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, galanin,motilin, nociceptin, endothelin, cAMP, adenosine, muscarinic,acetylcholine, serotonin, histamine, thrombin, kinin, folliclestimulating hormone, opsin, endothelial differentiation gene-1,rhodopsin, odorant, and cytomegalovirus receptors.

Most G-protein coupled receptors have single conserved cysteine residuesin each of the first two extracellular loops which form disulfide bondsthat are believed to stabilize functional protein structures. The 7transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6 andTM7. The cytoplasmic loop which connects TM5 and TM6 may be a majorcomponent of the G-protein binding domain.

Most G-protein coupled receptors contain potential phosphorylation siteswithin the third cytoplasmic loop and/or the carboxy terminus. Forseveral G-protein coupled receptors, such as the β-adrenoreceptor,phosphorylation by protein kinase A and/or specific receptor kinasesmediates receptor desensitization.

Recently, it was discovered that certain GPCRs, like thecalcitonin-receptor like receptor, might interact with small single passmembrane proteins called receptor activity modifying proteins (RAMP's).This interaction of the GPCR with a certain RAMP is determining whichnatural ligands have relevant affinity for the GPCR-RAMP combination andregulate the functional signaling activity of the complex (McLathie, L.M. et al., Nature (1998) 393:333-339).

For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise hydrophilic sockets formed by severalG-protein coupled receptor transmembrane domains, said sockets beingsurrounded by hydrophobic residues of the G-protein coupled receptors.The hydrophilic side of each G-protein coupled receptor transmembranehelix is postulated to face inward and form a polar ligand-binding site.TM3 has been implicated in several G-protein coupled receptors as havinga ligand-binding site, such as the TM3 aspartate residue. TM5 serines, aTM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331).Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshas been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

Receptors—primarily the GPCR class—have led to more than half of thecurrently known drugs (Drews, Nature Biotechnology, 1996, 14:1516). Thisindicates that these receptors have an established, proven history astherapeutic targets. Clearly there is a need for identification andcharacterization of further receptors which can play a role inpreventing, ameliorating or correcting dysfunctions or diseases,including, but not limited to PNS, psychiatric and CNS disorders,including schizophrenia, episodic paroxysmal anxiety (EPA) disorderssuch as obsessive compulsive, disorder (OCD), post traumatic stressdisorder (PTSD), phobia and panic, major depressive disorder, bipolardisorder, Parkinson's disease, general anxiety disorder, autism,delirium, multiple sclerosis, Alzheimer disease/dementia and otherneurodegenerative diseases, severe mental retardation, dyskinesias,Huntington's disease, Tourett's syndrome, tics, tremor, dystonia,spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleepdisorder, epilepsy, migraine; attention deficit/hyperactivity disorder(ADHD); cardiovascular diseases, including heart failure, anginapectoris, arrhythmias, myocardial infarction, cardiac hypertrophy,hypotension, hypertension—e.g. essential hypertension, renalhypertension, or pulmonary hypertension, thrombosis, arteriosclerosis,cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to IGS4 polypeptides (including theIGS4A and IGS4B polypeptide polymorphs), polynucleotides and recombinantmaterials and methods for their production. Another aspect of theinvention relates to methods for using such IGS4 polypeptides,polynucleotides and recombinant materials. Such uses include, but arenot limited to, use as a therapeutic target and for the treatment ofPNS, psychiatric and CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation, dyskinesias, Huntington's disease, Tourett's syndrome,tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders, among others. Preferred uses of theinvention relate to disorders of the nervous system, including thecentral nervous system (CNS) and the peripheral nervous system (PNS),disorders of the gastrointestinal system and/or of the cardiovascularsystem and/or of skeletal muscle and/or of the thyroid, and/or also tolung diseases, immunological diseases and disorders of the genitourinarysystem.

In still another aspect, the invention relates to methods to identifyagonists and antagonists using the materials provided by the invention,and treating conditions associated with IGS4 imbalance with theidentified compounds. Yet another aspect of the invention relates todiagnostic assays for detecting diseases associated with inappropriateIGS4 activity or levels. A further aspect of the invention relates toanimal-based systems which act as models for disorders arising fromaberrant expression or activity of IGS4. Preferred agonists orantagonists identified according to the present invention are thosewhich are suited for the treatment of disorders of the nervous system,including the central nervous system (CNS) and the peripheral nervoussystem (PNS), disorders of the gastrointestinal system and/or of thecardiovascular system and/or of skeletal muscle and/or of the thyroid,and/or also to lung diseases, immunological diseases and disorders ofthe genitourinary system.

The invention also relates to the identification of the cognate ligandof the IGS4 polypeptides of the invention. High affinity binding to saidIGS4 polypeptides is found for the neuropeptides known as neuromedin U.

TABLE 1 IGS4A-DNA of SEQ ID NO: 1 and SEQ ID NO: 3 5′-GGCTCAGCTTGAAACAGAGCCTCGTACCAGGGGAGGCTCAGGCCTTGGATTTTAATGTCAGGGATGGAAAAACTTCAGAATGCTTCCTGGATCTACCAGCAGAAACTAGAAGATCCATTCCAGAAACACCTGAACAGCACCGAGGAGTATCTGGCCTTCCTCTGCGGACCTCGGCGCAGCCACTTCTTCCTCCCCGTGTCTGTGGTGTATGTGCCAATTTTTGTGGTGGGGGTCATTGGCAATGTCCTGGTGTGCCTGGTGATTCTGCAGCACCAGGCTATGAAGACGCCCACCAACTACTACCTCTTCAGCCTGGCGGTCTCTGACCTCCTGGTCCTGCTCCTTGGAATGCCCCTGGAGGTCTATGAGATGTGGCGCAACTACCCTTTCTTGTTCGGGCCCGTGGGCTGCTACTTCAAGACGGCCCTCTTTGAGACCGTGTGCTTCGCCTCCATCCTCAGCATCACCACCGTCAGCGTGGAGCGCTACGTGGCCATCCTACACCCGTTCCGCGCCAAACTGCAGAGCACCCGGCGCCGGGCCCTCAGGATCCTCGGCATCGTCTGGGGCTTCTCCGTGCTCTTCTCCCTGCCCAACACCAGCATCCATGGCATCAAGTTCCACTACTTCCCCAATGGGTCCCTGGTCCCAGGTTCGGCCACCTGTACGGTCATCAAGCCCATGTGGATCTACAATTTCATCATCCAGGTCACCTCCTTCCTATTCTACCTCCTCCCCATGACTGTCATCAGTGTCCTCTACTACCTCATGGCACTCAGACTAAAGAAAGACAAATCTCTTGAGGCAGATGAAGGGAATGCAAATATTCAAAGACCCTGCAGAAAATCAGTCAACAAGATGCTGTTTGTCTTGGTCTTAGTGTTTGCTATCTGTTGGGCCCCGTTCCACATTGACCGACTCTTCTTCAGCTTTGTGGAGGAGTGGAGTGAATCCCTGGCTGCTGTGTTCAACCTCGTCCATGTGGTGTCAGGTGTCTTCTTCTACCTGAGCTCAGCTGTCAACCCCATTATCTATAACCTACTGTCTCGCCGCTTCCAGGCAGCATTCCAGAATGTGATCTCTTCTTTCCACAAACAGTGGCACTCCCAGCATGACCCACAGTTGCCACCTGCCCAGCGGAACATCTTCCTGACAGAATGCCACTTTGTGGAGCTGACCGAAGATATAGGTCCCCAATTCCCATGTCAGTCATCCATGCACAACTCTCACCTCCCAACAGCCCTCTCTAGTGAACAGATGTCAAGAACAAACTATCAAAGCTTCCACTTTAACAAAACCTGAATTCTTTCAGAGCTGACTCTCCTCTATGCCTCAAAACTTCAGAGAGGAACATCCCATAATGTATGCCTTCTCATATGATTATTAGAGAGGTAGATGGCTCTTACAACTCATGTACCCATTGCTAGTTTTTTTTTTTTAATAAACGTGAAAACTGAGAGTTAGATCTGGTTTCAAAACCCAAGACTGCCTGATTTTTAGTTATCTTTCCACTATCCTAACTGCCTCATGCCCCTTCACTAGTTCATGCCAAGAACGTGACTGGAAAGGCATGGCACCTATACCTTGATTAATTTCCATTAATGGAAATGGTTCGTCCTGAGTCATCTACGTTCCGAGTCAGGCTGTCAC TCCTACTA-3′

TABLE 2 IGS4B-DNA of SEQ ID NO: 5 and SEQ ID NO: 7 5′-GGCTCAGCTTGAAACAGAGCCTCGTACCAGGGGAGGCTCAGGCCTTGGATTTTAATGTCAGGGATGGAAAAACTTCAGAATGCTTCCTGGATCTACCAGCAGAAACTAGAAGATCCATTCCAGAAACACCTGAACAGCACCGAGGAGTATCTGGCCTTCCTCTGCGGACCTCGGCGCAGCCACTTCTTCCTCCCCGTGTCTGTGGTGTATGTGCCAATTTTTGTGGTGGGGGTCATTGGCAATGTCCTGGTGTGCCTGGTGATTCTGCAGCACCAGGCTATGAAGACGCCCACCAACTACTACCTCTTCAGCCTGGCGGTCTCTGACCTCCTGGTCCTGCTCCTTGGAATGCCCCTGGAGGTCTATGAGATGTGGCGCAACTACCCTTTCTTGTTCGGGCCCGTGGGCTGCTACTTCAAGACGGCCCTCTTTGAGACCGTGTGCTTCGCCTCCATCCTCAGCATCACCACCGTCAGCGTGGAGCGCTACGTGGCCATCCTACACCCGTTCCGCGCCAAACTGCAGAGCACCCGGCGCCGGGCCCTCAGGATCCTCGGCATCGTCTGGGGCTTCTCCGTGCTCTTCTCCCTGCCCAACACCAGCATCCATGGCATCAAGTTCCACTACTTCCCCAATGGGTCCCTGGTCCCAGGTTCGGCCACCTGTACGGTCATCAAGCCCATGTGGATCTACAATTTCATCATCCAGGTCACCTCCTTCCTATTCTACCTCCTCCCCATGACTGTCATCAGTGTCCTCTACTACCTCATGGCACTCAGACTAAAGAAAGACAAATCTCTTGAGGCAGATGAAGGGAATGCAAATATTCAAAGACCCTGCAGAAAATCAGTCAACAAGATGCTGTTTGTCTTGGTCTTAGTGTTTGCTATCTGTTGGGCCCCGTTCCACATTGACCGACTCTTCTTCAGCTTTGTGGAGGAGTGGACTGAATCCCTGGCTGCTGTGTTCAACCTCGTCCATGTGGTGTCAGGTGTCTTATTCTACCTGAGCTCAGCTGTCAACCCCATTATCTATAACCTACTGTCTCGCCGCTTCCAGGCAGCATTCCAGAATGTGATCTCTTCTTTCCACAAACAGTGGCACTCCCAGCATGACCCACAGTTGCCACCTGCCCAGCGGAACATCTTCCTGACAGAATGCCACTTTGTGGAGCTGACCGAAGATATAGGTCCCCAATTCCTATGTCAGTCATCCGTGCACAACTCTCACCTCCCAACAGCCCTCTCTAGTGAACAGATGTCAAGAACAAACTATCAAAGCTTCCACTTTAACAAAACCTGAATTCTTTCAGAGCTGACTCTCCTCTATGCCTCAAAACTTCAGAGAGGAACATCCCATAATGTATGCCTTCTCATATGAAATTAGAGAGGTAGAATGGCTCTTACAACTCATGTACCCATTGCTAGTTTTTTTTTTTTAATAAACGTGAAAACTGAGAGTTAGATCTGGTTTCAAAACCCAAGACTGCCTGATTTTTAGTTATCTTTCCACTATCCTAACTGCCTCATGCCCCTTCACTAGTTCATGCCAAGAACGTGACTGGAAAGGCATGGCACCTATACCTTGATTAATTTCCATTAATGGAAATGGTTCGTCCTGAGTCATCTACGTTCCGAGTCAGGCTGTCAC TCCTACTA-3′

TABLE 3 IGS4A-64-DNA of SEQ ID NO: 9 and SEQ ID NO: 11 5′-GGCTCAGCTTGAAACAGAGCCTCGTACCAGGGGAGGCTCAGGCCTTGGATTTTAATGTCAGGGATGGAAAAACTTCAGAATGCTTCCTGGATCTACCAGCAGAAACTAGAAGATCCATTCCAGAAACACCTGAACAGCACCGAGGAGTATCTGGCCTTCCTCTGCGGACCTCGGCGCAGCCACTTCTTCCTCCCCGTGTCTGTGGTGTATGTGCCAATTTTTGTGGTGGGGGTCATTGGCAATGTCCTGGTGTGCCTGGTGATTCTGCAGCACCAGGCTATGAAGACGCCCACCAACTACTACCTCTTCAGCCTGGCGGTCTCTGACCTCCTGGTCCTGCTCCTTGGAATGCCCCTGGAGGTCTATGAGATGTGGCGCAACTACCCTTTCTTGTTCGGGCCCGTGGGCTGCTACTTCAAGACGGCCCTCTTTGAGACCGTGTGCTTCGCCTCCATCCTCAGCATCACCACCGTCAGCGTGGAGCGCTACGTGGCCATCCTACACCCGTTCCGCGCCAAACTGCAGAGCACCCGGCGCCGGGCCCTCAGGATCCTCGGCATCGTCTGGGGCTTCTCCGTGCTCTTCTCCCTGCCCAACACCAGCATCCATGGCATCAAGTTCCACTACTTCCCCAATGGGTCCCTGGTCCCAGGTTCGGCCACCTGTACGGTCATCAAGCCCATGTGGATCTACAATTTCATCATCCAGGTCACCTCCTTCCTATTCTACCTCCTCCCCATGACTGTCATCAGTGTCCTCTACTACCTCATGGCACTCAGACTAAAGAAAGACAAATCTCTTGAGGCAGATGAAGGGAATGCAAATATTCAAAGACCCTGCAGAAAATCAGTCAACAAGATGCTGTCTTTGTGGAGGAGTGGAGTGAATCCCTGGCTGCTGTGTTCAACCTCGTCCATGTGGTGTCAGGTGTCTTCTTCTACCTGAGCTCAGCTGTCAACCCCATTATCTATAACCTACTGTCTCGCCGCTTCCAGGCAGCATTCCAGAATGTGATCTCTTCTTTCCACAAACAGTGGCACTCCCAGCATGACCCACAGTTGCCACCTGCCCAGCGGAACATCTTCCTGACAGAATGCCACTTTGTGGAGCTGACCGAAGATATAGGTCCCCAATTCCCATGTCAGTCATCCATGCACAACTCTCACCTCCCAACAGCCCTCTCTAGTGAACAGATGTCAAGAACAAACTATCAAAGCTTCCACTTTAACAAAACCTGAATTCTTTCAGAGCTGACTCTCCTCTATGCCTCAAAACTTCAGAGAGGAACATCCCATAATGTATGCCTTCTCATATGATATTAGAGAGGTAGAATGGCTCTTACAACTCATGTACCCATTGCTAGTTTTTTTTTTTTAATAAACGTGAAAACTGAGAGTTAGATCTGGTTTCAAAACCCAAGACTGCCTGATTTTTAGTTATCTTTCCACTATCCTAACTGCCTCATGCCCCTTCACTAGTTCATGCCAAGAACGTGACTGGAAAGGCATGGCACCTATACCTTGATTAATTTCCATTAATGGAAATGGTTCGTCCTGAGTCATCTACGTTCCGAGTCAGGCTGTCACTCCTACTA-3′

TABLE 4 IGS4A-protein of SEQ ID NO: 2 and SEQ ID NO: 4 (without thethree amino acids between brackets). (MSG)MEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPTNYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRLKKDKSLEADEGNANIQRPCRKSVNKMLFVLVLVFAICWAPFHIDRLFFSFVEEWSESLAAVFNLVHVVSGVFFYLSSAVNPIIYNLLSRRFQAAFQNVISSFHKQWHSQHDPQLPPAQRNIFLTECHFVELTEDIGPQFPCQSSMHNSHLPTAL SSEQMSRTNYQSFHFNKT

TABLE 5 IGS4B-protein of SEQ ID NO: 6 and SEQ ID NO: 8 (without thethree amino acids between brackets). (MSG)MEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPTNYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRLKKDKSLEADEGNANIQRPCRKSVNKMLFVLVLVFAICWAPFHIDRLFFSFEEWTESLAAVFNLVHVVSGVLFYLSSAVNPIIYNLLSRRFQAAFQNVISSFHKQWHSQHDPQLPPAQRNIFLTECHFVELTEDIGPQFLCQSSVHNSHLPTAL SSEQMSRTNYQSFHFNKT

TABLE 6 IGS4A-64-protein of SEQ ID NO: 10 and SEQ ID NO: 12 (withoutthree amino acids between brackets). (MSG)MEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPTNYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRLKKDKSLEADEGNANIQRPCRKSVNKMLSLWRSGVNPWLLCSTSSMWCQVSSST

DESCRIPTION OF THE INVENTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“IGS4” refers, among others, to a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 (IGS4A) and SEQ IDNO: 6 or SEQ ID NO: 8 (IGS4B), or a variant thereof. Particularlypreferred are polypeptides of IGS4B.

“Receptor Activity” or “Biological Activity of the Receptor” refers tothe metabolic or physiologic function of said IGS4 including similaractivities or improved activities or these activities with decreasedundesirable side effects. Also included are antigenic and immunogenicactivities of said IGS4.

“IGS4-gene” refers to a polynucleotide comprising the nucleotidesequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ IDNO: 7 or variants thereof and/or their complements.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of a Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural stateand/or separated from the natural environment. Thus, if an “isolated”composition or substance that occurs in nature has been “isolated”, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated”, but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis a mixture of single-and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” may also include triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The term polynucleotide alsoincludes DNAs or RNAs containing one or more modified bases and DNAs orRNAs with backbones modified for stability or for other reasons.“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications has been made to DNAand RNA; thus, “polynucleotide” embraces chemically, enzymatically ormetabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells. “Polynucleotide” also embraces relatively shortpolynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to short chains,commonly referred to as peptides, oligopeptides or oligomers, and tolonger chains, generally referred to as proteins, and/or to combinationsthereof. Polypeptides may contain amino acids other than the 20gene-encoded amino acids. “Polypeptides” include amino acid sequencesmodified either by natural processes, such as posttranslationalprocessing, or by chemical modification techniques which are well knownin the art. Such modifications are well-described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature. Modifications can occur anywhere in a polypeptide, includingthe peptide backbone, the amino acid side-chains and the amino orcarboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Polypeptides may be branched as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol; cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. See, for instance,PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993 and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York, 1983; Seifter et al., “Analysisfor protein modifications and nonprotein cofactors”, Meth. Enzymol.(1990) 182:626-646 and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging”, Ann. NY Acad. Sci. (1992)663:48-62.

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties such as essentialbiological, structural, regulatory or biochemical propeties. A typicalvariant of a polynucleotide differs in nucleotide sequence from another,reference polynucleotide. Changes in the nucleotide sequence of thevariant may or may not alter the amino acid sequence of a polypeptideencoded by the reference polynucleotide. Nucleotide changes may resultin amino acid substitutions, additions, deletions, fusions andtruncations in the polypeptide encoded by the reference sequence, asdiscussed below. A typical variant of a polypeptide differs in aminoacid sequence from another, reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more substitutions, additions, anddeletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed.; Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J. AppliedMath. (1988) 48:1073). Methods commonly employed to determine identityor similarity between two sequences include, but are not limited to,those disclosed in Guide to Huge Computers, Martin J. Bishop, ed.,Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAMJ. Applied Math. (1988) 48:1073. Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, GCG program package (Devereux, J., etal., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA(Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403). The word“homology” may substitute for the word “identity”.

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO: 1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five nucleotide differencesper each 100 nucleotides of the reference nucleotide sequence of SEQ IDNO: 1. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto any 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up toany 5% of the total nucleotides in the reference sequence may beinserted into the reference sequence, or in a number of nucleotides ofup to any 5% of the total nucleotides in the reference sequence theremay be a combination of deletion, insertion and substitution. Thesemutations of the reference sequence may occur at the 5 or 3 terminalpositions of the reference nucleotide sequence or anywhere between thoseterminal positions, interspersed either individually among nucleotidesin the reference sequence or in one or more contiguous groups within thereference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO: 2 is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO: 2. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to any 5% of the aminoacid residues in the reference sequence may be deleted or substitutedwith another amino acid, or a number of amino acids up to any 5% of thetotal amino acid residues in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to IGS4 polypeptides (orIGS4 proteins). The IGS4 polypeptides include the polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 and the polypeptidehaving the amino acid sequence encoded by the DNA insert contained inthe deposit no. CBS102221 or deposit no. CBS102222, deposited on Sep.24, 1999 at the Centraalbureau voor Schimmelcultures at Baarn theNetherlands; as well as polypeptides comprising the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and thepolypeptide having the amino acid sequence encoded by the DNA insertcontained in the deposit no. CBS102221 or deposit no. CBS102222 at theCentraalbureau voor Schimmelcultures at Baarn the Netherlands andpolypeptides comprising the amino acid sequence which have at least 80%identity to that of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ IDNO: 8 and/or the polypeptide having the amino acid sequence encoded bythe DNA insert contained in the deposit no. CBS102221 or deposit no.CBS102222 at the Centraalbureau voor Schimmelcultures at Baarn theNetherlands over its entire length, and still more preferably at least90% identity, and even still more preferably at least 95% identity tosaid amino acid sequences. Furthermore, those with at least 97%, inparticular at least 99%, are highly preferred. Also included within IGS4polypeptides are polypeptides having the amino acid sequence which hasat least 80% identity to the polypeptide having the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 or thepolypeptide having the amino acid sequence encoded by the DNA insertcontained in the deposit no. CBS102221 or deposit no. CBS102222 at theCentraalbureau voor Schimmelcultures at Baarn the Netherlands over itsentire length, and still more preferably at least 90% identity, and evenstill more preferably at least 95% identity to SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6 or SEQ ID NO: 8. Furthermore, those with at least 97%,in particular at least 99% are highly preferred. Preferably IGS4polypeptides exhibit at least one biological activity of the receptor.

In an additional embodiment of the invention, the IGS4 polypeptides maybe a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, sequences which aid indetection such as antigenic peptide tags (such as the haemagglutinin(HA) tag), or an additional sequence for stability during recombinantproduction.

Fragments of the IGS4 polypeptides are also included in the invention. Afragment is a polypeptide having an amino acid sequence that is the sameas part of, but not all of, the amino acid sequence of theaforementioned IGS4 polypeptides. As with IGS4 polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20; 21-40, 41-60, 61-80, 81-100; and 101 to the end of IGS4polypeptide. In this context “about” includes the particularly recitedranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of IGS4 polypeptides, except for deletion of acontinuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. An example of atruncated polypeptide according to the present invention is thepolypetide of SEQ ID NO: 10 and SEQ ID NO: 12, which is encoded by thepolynucleotide of SEQ ID NO: 9 respectively SEQ ID NO: 11. Alsopreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions. Otherpreferred fragments are biologically active fragments. Biologicallyactive fragments are those that mediate receptor activity, includingthose with a similar activity or an improved activity, or with adecreased undesirable activity. Also included are those that areantigenic or immunogenic in an animal, especially in a human.

Thus, the polypeptides of the invention include polypeptides having anamino acid sequence at least 80% identical to that of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and/or the polypeptide having theamino acid sequence encoded by the DNA insert contained in the depositno. CBS102221 or the deposit no. CBS102222 at the Centraalbureau voorSchimmelcultures at Baarn the Netherlands, or fragments thereof with atleast 80% identity to the corresponding fragment. Preferably, all ofthese polypeptide fragments retain the biological activity of thereceptor, including antigenic activity. Variants of the defined sequenceand fragments also form part of the present invention. Preferredvariants are those that vary from the referents by conservative aminoacid substitutions—i.e., those that substitute a residue with another oflike characteristics. Typical such substitutions are among Ala, Val, Leuand Ile; among Ser and Thr; among the acidic residues Asp and Glu; amongAsn and Gln; and among the basic residues Lys and Arg; or aromaticresidues Phe and Tyr. Particularly preferred are variants in whichseveral, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, oradded in any combination.

With regard to the polypeptides of the present invention it was alsofound that they show a high affinity binding for neuromedin U, inparticular for neuromedin U-8 (an oligopeptide of 8 amino acids),neuromedin U-23 (an oligopeptide of 23 amino acids) and/or neuromedinU-25 (an oligopeptide of 25 amino acids). In the context of the presentinvention the term “high affinity” is understood as to describe a ligandbinding showing log EC₅₀ values of at least below −6.00 (approx. 660nM), preferably log EC₅₀ below −7.00 (approx. 55 nM), more preferablylog EC₅₀ below −9.00 (approx. 500 pM to 1.2 nM), and most preferably logEC₅₀ below −10.00 (approx. 50-100 pM).

Two forms of the neuropeptide neuromedin U, neuromedin U-8 andneuromedin U-25, are described in the literature as uterus stimulatingand hypertensive peptides (Minamino et al., 1985, Biochem. Biophys. Res.Commun. 130:1078-1085) being originally isolated from the porcine spinalcord. For neuromedin U-23, an oligopeptide of 23 amino acids, see forexample: Okimura et al., Pept. Chem. (1995), Vol. Date 1994, 32:321-324;Salmon et al., J. Biol. Chem. (2000), 275(7), 4549-4554. Neuromedin U(NMU) was subsequently isolated from a number of species, e.g. from rat(NMU-23), human (NMU-25), frog (NMU-25), dog (NMU-8 and NMU-25), rabbit(NMU-25), and chicken (NMU-25). Thus, Domin et al. described thecharacterization of neuromedin U like immunoreactivity in rat, porcine,guinea pig and human tissue extracts using a specific radioimmunoassay(1986, Biochem. Biophys. Res. Commun. 140:1127-34). The primarystructure of neuromedin U 23 from the rat ileum was established byConlon et al. (1988, J. Neurochem. 51:988-991). Minamino et al. (1988,Biochem. Biophys. Res. Commun. 156:355-360) have isolated rat neuromedinU from the small intestine using mainly immunoaffinity chromatographyand radioimmunoassay for pig neuromedin U-8, and the amino acid sequenceof rat neuromedin U was determined by microsequence analysis and thestructure was confirmed by synthesis. Although the C-terminalheptapeptide amide structure of pig neuromedin U is completely conservedin rat neuromedin U. the remainder of the peptide reveals nine aminoacid replacements and two amino acid deletions when compared to pigneuromedin U-25. The distribution, primary structure, and relativebiological activity of neuromedin U has been determined also in the frogRana temporaria by Domin et al. (1989, J. Biol. Chem. 264:20881-20885)showing that the entire sequence was found to be an icosapentapeptidewhich displays marked sequence similarity to both porcine and ratneuromedin U. In a further study Domin et al. (1992, Regul. Pept.41:1-8) have purified an avian homolog of neuromedin U from the chicken.Microsequence analysis characterized the peptide to be 25 amino acidresidues long, and chicken neuromedin U showed marked sequencesimilarity with the porcine peptide at its bioactive C-terminal region.Isolation, structural characterization and pharmacological activity ofdog neuromedin U-25 was described by O'Harte et al. (1991 Peptides12:11-15). Furthermore, for rabbit neuromedin U-25 it was found that itlacks conservation of a posttranslational processing site (Kage et al.,1991 Regul. Pept. 33:191-198); thus, in rabbit neuromedin U, theArg16-Arg17 dibasic residue processing site that is found in pig and dogneuromedin U-25 is replaced by Arg-Gly, but this potential monobasicprocessing site is not utilized by cleavage enzyme(s) in the intestirie.

Among the species studied the 5 amino acids at the C-terminus of thepeptide were found to be almost totally conserved, suggesting that thisregion is of major importance. Thus, mammalian neuromedins share acommon C-terminal sequence “-Phe-Leu-Phe-Arg-Pro-Arg-Asn-amide” [SEQ IDNO: 35] which appears to be essential for its biological activities. NMUis distributed both in the gastrointestinal tract and the centralnervous System (CNS). In the rat, the highest concentration ofneuromedin (NMU) was found in the ileum, followed by the pituitary,hypothalamus, spinal cord, thyroid, and the genitourinary tract.Immunohistochemistry studies showed that NMU immunoreactivity in the gutwas only found in nerve fibers, mainly in the myenteric and submucousplexuses, and in the mucosa of all areas except stomach while no NMUimmunoreactivity was found in endocrine cells. In the rat brain, NMUimmunoreactivity was found in fibers widespread throughout the brainwith the exception of the cerebellum. Human and rat genes encoding NMUprecursor have been isolated. Both encode NMU at the C-terminus andother potential peptide products in the middle (Lo et al., 1992, J. Mol.Endocrinol. 6:1538-1544; Austin et al., 1995, J. Mol. Endocrinol.14:157-169). High affinity NMU binding was characterized in rat uterus,and was shown to be sensitive to GTP- -S (Nandha et al., 1993,Endocrinology 133:482-486), suggesting that a receptor for NMU should bea G-protein coupled receptor. Nevertheless, the physiological role ofNMU remains largely unknown. Neuromedin U (NMU) can cause potentcontraction of smooth muscle, increase arterial blood pressure, modifyintestinal ion transport, and at low doses stimulates the function andgrowth of the adrenal cortex. NMU was also shown to reduce the bloodflow in superior enteric artery and portal vein while increasing bloodflow slightly in pancreatic tissue.

Furthermore, according to the international patent application WO90/01330 the neuromedins U-8 and U-25 are described to be suitable inthe treatment of disorders of the gastrointestinal tract, e.g. beinguseful in the selective reduction of blood flow to the gastrointestinaltract, in the treatment of gastrointestinal bleeding and postprandialhypotension.

The IGS4 polypeptides of the present invention have been identified as aG-protein coupled receptor responsive to neuromedin U or ligandssufficiently similar thereto. Thus the IGS4 receptor, in particular theIGS4B receptor, responsive to neuromedin U will greatly facilitate theunderstanding of the physiological mechanisms of neuromedin U and otherligands sufficiently similar thereto, as well as of related diseases.

The tissue distribution of the polypeptides of the present invention andthe expression levels are shown in the FIGS. 5-8, from which the skilledartisan can estimate the localisation and relevance of expression. Forinstance, with regard to the tissue distribution of the polypeptides ofthe present invention it was found, e.g. by MTE (multiple tissueexpression) analysis, Northern blot analysis and Quantitative RT-PCRexpression analysis that the IGS4 polypeptides of the present inventionparticularly are brought to expression with a medium level (relative toexpression in testis as 100% in MTE blot, or in spinal cord as 100% inQuantitative RT-PCR analysis, respectively) e.g. in brain, skeletalmuscle, cerebellum, thymus, medulla, thyroid, trachea, thalamus,substantia nigra, corpus callosum, caudate nucleus, pons, nucleusaccumbens, fetal brain and stomach; and with a relevant level (if beingdetectable by Quantitative RT-PCR analysis) e.g. in heart, lung, andprostate. For instance, expression levels are considered to be medium ifthey amount at least 20% of the expression value found for the by farhighest expression (set as 100%) in testis or spinal cord. For instance,expression levels are considered to be relevant if expression could bedetected at least via Quantitative RT-PCR analysis. It will beappreciated that expression levels indicated for any organ are averagevalues of expression levels in the specific tissues and cell typesconstituting the organ. Thus, if an expression level is just found to berelevant with respect to an organ, this does not necessarily excludemedium or even high expression levels locally within a specific region,e.g. in a specific tissue and/or cell type, of the organ.

These results suggest that IGS4 polypeptides preferably play a role inthe nervous system, including the central nervous system (CNS) and theperipheral nervous system (PNS), in the gastrointestinal system and/orin the cardiovascular system and/or in skeletal muscle and/or in thethyroid, and/or also in lung diseases, immunological diseases anddisorders of the genitourinary system.

Thus, in a further embodiment the invention pertains also to an isolatedIGS4 polypeptide comprising an amino acid sequence of a neuromedinreceptor protein, preferably of a mammalian neuromedin receptor protein,said protein exhibiting high affinity binding for neuromedin U,preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedinU-25. Particularly, the isolated IGS4 polypeptide comprising an aminoacid sequence of a neuromedin receptor protein, is a protein exhibitingexpression (being at least detectable via Northern and/or MTE and/orQuantitative RT-PCR analysis) in brain, skeletal muscle, cerebellum,testis, corpus callosum, spinal cord, substantia nigra, medulla,thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain,stomach, heart, thyroid gland, lung, thymus, prostate and/or in trachea.In a variant of this embodiment the invention pertains to an isolatedIGS4 polypeptide comprising an amino acid sequence of a neuromedinreceptor protein, preferably of a mammalian neuromedin receptor protein,said protein exhibiting high affinity binding for neuromedin U,preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedinU-25, said protein exhibiting expression (being at least detectable viaNorthern and/or MTE and/or Quantitative RT-PCR analysis) in brain,skeletal muscle, cerebellum, testis, corpus callosum, spinal cord,substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleusaccumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus,prostate and/or in trachea, and said amino acid sequence being selectedfrom the group of amino acid sequence as already defined supra.

The IGS4 polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Methods for preparing such polypeptides are well known inthe art.

Polynucleotides of the Invention

A further aspect of the invention relates to IGS4 polynucleotides. IGS4polynucleotides include isolated polynucleotides which encode the IGS4polypeptides (including IGS4A and IGS4B) and fragments, andpolynucleotides closely related thereto. More specifically, the IGS4polynucleotide of the invention includes a polynucleotide comprising thenucleotide sequence contained in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5 or SEQ ID NO: 7 encoding a IGS4A polypeptide of SEQ ID NO: 2 or of SEQID NO: 4 and a IGS4B polypeptide of SEQ ID NO: 6 or of SEQ ID NO: 8respectively, polynucleotides having the particular sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 and polynucleotideswhich essentially correspond to the DNA insert contained in the depositno. CBS102221 or the deposit no. CBS102222 at the Centraalbureau voorSchimmelcultures at Baarn the Netherlands.

IGS4 polynucleotides further include polynucleotides comprising anucleotide sequence that has at least 80% identity over its entirelength to a nucleotide sequence encoding the IGS4 polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, a polynucleotidecomprising a nucleotide sequence that is at least 80% identical to thatof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 over itsentire length and a polynucleotide which essentially correspond to theDNA insert contained in the deposit no. CBS102221 or the deposit no.CBS102222 at the Centraalbureau voor Schimmelcultures at Baarn theNetherlands.

In this regard, polynucleotides with at least 90% identity areparticularly preferred, and those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferred andthose with at least 98-99% are most highly preferred, with at least 99%being the most preferred. Also included under IGS4 polynucleotides are anucleotide sequence which has sufficient identity to a nucleotidesequence contained in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ IDNO: 7 or to the DNA insert contained in the deposit no. CBS102221 or inthe deposit no. CBS102222 at the Centraalbureau voor Schimmelcultures atBaarn the Netherlands to hybridize under conditions useable foramplification or for use as a probe or marker. The invention alsoprovides polynucleotides which are complementary to such IGS4polynucleotides.

IGS4 of the invention is structurally related to other proteins of theG-protein coupled receptor family, as shown by the results of BLASTsearches in the public databases. The amino acid sequence of Table 1(SEQ ID NO: 2) has about 46% identity (using BLAST, Altschul S. F. etal. [1997], Nucleic Acids Res. 25:3389-3402) over most of its length(316 amino acid residues) with a human orphan G-protein coupled receptor(Accession # O43664, Tan et al., Genomics 52(2):223-229 (1998). There is27% homology (over amino acid residues 61-349) to the rat neurotensin 1receptor (Accession # P20789 Tanaka K. et al, Neuron 4:847-854 (1990)).The nucleotide sequence of Table 1 (SEQ ID NO: 1) is 63% identical to anorphan G-protein coupled receptor over nucleotide residues 120-864(Accession # AF044600, corresponding with the protein sequence 043664).Furthermore, there is 53% identity to the human growth hormonesecretagogue receptor over residues 94-1137 (Howard A. D. et al, Science273:974-977(1996)). Thus, IGS4 polypeptides and polynucleotides of thepresent invention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides, and their utility is obvious to anyone skilled in theart.

Polynucleotides of the invention can be obtained from natural sourcessuch as genomic DNA. In particular, degenerated PCR primers can bedesigned that encode conserved regions within a particular GPCR genesubfamily. PCR amplification reactions on genomic DNA or cDNA using thedegenerate primers will result in the amplification of several members(both known and novel) of the gene family under consideration (thedegenerated primers must be located within the same exon, when a genomictemplate is used). (Libert et al., Science, 1989, 244: 569-572).Polynucleotides of the invention can also be synthesized usingwell-known and commercially available techniques (e.g. F. M. Ausubel etal, 2000, Current Protocols in Molecular Biology).

The nucleotide sequence encoding the IGS4 polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 may be identical to thepolypeptide encoding sequence contained in SEQ ID NO: 1 (nucleotidenumber 55 to 1299) or SEQ ID NO: 3 (nucleotide number 64 to 1299), orSEQ ID NO: 5 (nucleotide number 55 to 1299) or SEQ ID NO: 7 (nucleotidenumber 64 to 1299) respectively, or it may be a different nucleotidesequence, which as a result of the redundancy (degeneracy) of thegenetic code might also show alterations compared to the polypeptideencoding sequence contained in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5or SEQ ID NO: 7, but also encodes the polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, respectively.

In a further embodiment the invention pertains to an isolated nucleotidesequence encoding an IGS4 neuromedin receptor protein, preferablyencoding a mammalian neuromedin receptor protein, said proteinexhibiting high affinity binding for neuromedin U, preferably forneuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25.Particularly, the isolated nucleotide sequence encodes an IGS4 euromedinreceptor protein which is a protein exhibiting expression (being atleast detectable via Northern and/or MTE and/or Quantitative RT-PCRanalysis) in brain, skeletal muscle, cerebellum, testis, corpuscallosum, spinal cord, substantia nigra, medulla, thalamus, caudatenucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroidgland, lung, thymus, prostate and/or in trachea. In a variant of thisembodiment the invention pertains to an isolated nucleotide sequenceencoding an IGS4 neuromedin receptor protein, preferably encoding amammalian neuromedin receptor protein, said protein exhibiting highaffinity binding for neuromedin U, preferably for neuromedin U-8, forneuromedin U-23 and/or for neuromedin U-25, said protein exhibitingexpression (being at least detectable via Northern and/or MTE and/orQuantitative RT-PCR analysis) in brain, skeletal muscle, cerebellum,testis, corpus callosum, spinal cord, substantia nigra, medulla,thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain,stomach, heart, thyroid gland, lung, thymus, prostate and/or in trachea,and said nucleotide sequence being selected from the group of nucleotidesequences as already defined supra.

When the polynucleotides of the invention are used for the recombinantproduction of the IGS4 polypeptide, the polynucleotide may include thecoding sequence for the mature polypeptide or a fragment thereof, byitself; the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro- proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci USA (1989) 86:821-824, or is an HA tag. Thepolynucleotide may also contain non-coding 5′ and 3′ sequences, such astranscribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding IGS4 variantscomprising the amino acid sequence of the IGS4 polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 in which several, 5-10,1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted oradded, in any combination.

The polynucleotides of the invention can be engineered using methodsgenerally known in the art in order to alter IGS4-encoding sequences fora variety of purposes including, but not limited to, modification of thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, oligonucleotide-mediated site-directedmutagenesis may be used to introduce mutations that create amino acidsubstitutions, create new restriction sites, alter modification (e.g.glycosylation or phosphorylation) patterns, change codon preference,produce splice variants, and so forth.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the polynucleotides described above. As hereinused, the term “stringent conditions” means hybridization will occuronly if there is at least 80%, and preferably at least 90%, and morepreferably at least 95%, yet even more preferably at least 97%, inparticular at least 99% identity between the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 or SEQ ID NO: 7 or a fragment thereof, may be used ashybridization probes for cDNA and genomic DNA, to isolate full-lengthcDNAs and genomic clones encoding IGS4 and to isolate cDNA and genomicclones of other genes (including genes encoding homologs and orthologsfrom species other than human) that have a high sequence similarity tothe IGS4 gene. People skilled in the art are well aware of suchhybridization techniques. Typically these nucleotide sequences are 80%identical, preferably 90% identical, more preferably 95% identical tothat of the referent. The probes generally will comprise at least 5nucleotides, and preferably at least 8 nucleotides, and more preferablyat least 10 nucleotides, yet even more preferably at least 12nucleotides, in particular at least 15 nucleotides. Most preferred, suchprobes will have at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will range between 30 and 50nucleotides.

One embodiment, to obtain a polynucleotide encoding the IGS4polypeptide, including homologs and orthologs from species other thanhuman, comprises the steps of screening an appropriate library understringent hybridization conditions with a labeled probe having the SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or a fragmentthereof, and isolating full-length cDNA and genomic clones containingsaid polynucleotide sequence. Such hybridization techniques are wellknown to those of skill in the art. Stringent hybridization conditionsare as defined above or alternatively conditions under overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate (w/v), and 20 microgram/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beused as research reagents and materials for discovery of treatments anddiagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be used to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals, i.e.derived from a different species.

If the IGS4 polypeptide is to be expressed for use in screening assays,generally, it is preferred that the polypeptide be produced at thesurface of the cell. In this event, the cells may be harvested prior touse in the screening assay. In case the affinity or functional activityof the IGS4 polypeptide is modified by receptor activity modifyingproteins (RAMP), coexpression of the relevant RAMP most likely at thesurface of the cell is preferred and often required. Also in this eventharvesting of cells expressing the IGS4 polypeptide and the relevantRAMP prior to use in screening assays is required. If the IGS4polypeptide is secreted into the medium, the medium can be recovered inorder to recover and purify the polypeptide; if producedintracellularly, the cells must first be lysed before the polypeptide isrecovered. Membranes expressing the IGS4 polypeptide can be recovered bymethods that are well known to a person skilled in the art. In general,such methods include harvesting of the cells expressing the IGS4polypeptide and homogenization of the cells by a method such as, but notlimited to, pottering. The membranes may be recovered by washing thesuspension one or several times.

IGS4 polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well-known techniques for refoldingproteins may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of IGS4 polynucleotides for useas diagnostic reagents. Detection of a mutated form of the IGS4 geneassociated with a dysfunction will provide a diagnostic tool that canadd to or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression, over-expression or alteredexpression of IGS4. Also in this event co-expression of relevantreceptor activity modifying proteins can be required to obtaindiagnostic assays of desired quality. Individuals carrying mutations inthe IGS4 gene may be detected at the DNA level by a variety oftechniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled IGS4 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science (1985) 230:1242. Sequence changes at specific locations may alsobe revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method. See Cotton et al., Proc.Natl. Acad. Sci. USA (1985) 85: 4397-4401. In another embodiment, anarray of oligonucleotide probes comprising the IGS4 nucleotide sequenceor fragments thereof can be constructed to conduct efficient screeningof e.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability. (See for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to PNS, psychiatric and CNS disorders, includingschizophrenia, episodic paroxysmal anxiety (EPA) disorders such asobsessive compulsive disorder (OCD), post traumatic stress disorder(PTSD), phobia and panic, major depressive disorder, bipolar disorder,Parkinson's disease, general anxiety disorder, autism, delirium,multiple sclerosis, Alzheimer disease/dementia and otherneurodegenerative diseases, severe mental retardation, dyskinesias,Huntington's disease, Tourett's syndrome, tics, tremor, dystonia,spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleepdisorder, epilepsy, migraine; attention deficit/hyperactivity disorder(ADHD); cardiovascular diseases, including heart failure, anginapectoris, arrhythmias, myocardial infarction, cardiac hypertrophy,hypotension, hypertension—e.g. essential hypertension. renalhypertension, or pulmonary hypertension, thrombosis, arteriosclerosis,cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders, through detection of mutation in the IGS4gene by the methods described. According to the present invention, thediagnostic assays offer in particular a process for diagnosing ordetermining a susceptibility to disorders of the nervous system,including the central nervous system (CNS) and the peripheral nervoussystem (PNS), disorders of the gastrointestinal system and/or of thecardiovascular system and/or of skeletal muscle and/or of the thyroid,and/or also to lung diseases, immunological diseases and disorders ofthe genitourinary system.

In addition, PNS, psychiatric and CNS disorders, includingschizophrenia, episodic paroxysmal anxiety (EPA) disorders such asobsessive compulsive disorder (OCD), post traumatic stress disorder(PTSD), phobia and panic, major depressive disorder, bipolar disorder,Parkinson's disease, general anxiety disorder, autism, delirium,multiple sclerosis, Alzheimer disease/dementia and otherneurodegenerative diseases, severe mental retardation, dyskinesias,Huntington's disease, Tourett's syndrome, tics, tremor, dystonia,spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleepdisorder, epilepsy, migraine; attention deficit/hyperactivity disorder(ADHD); cardiovascular diseases, including heart failure, anginapectoris, arrhythmias, myocardial infarction, cardiac hypertrophy,hypotension, hypertension—e.g. essential hypertension, renalhypertension, or pulmonary hypertension, thrombosis, arteriosclerosis,cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders, can be diagnosed by methods comprisingdetermining from a sample derived from a subject an abnormally decreasedor increased level of the IGS4 polypeptide or IGS4 mRNA. In particulardisorders of the nervous system, including the central nervous system(CNS) and the peripheral nervous system (PNS), disorders of thegastrointestinal system and/or of the cardiovascular system and/or ofskeletal muscle and/or of the thyroid, and/or also lung diseases,immunological diseases and disorders of the genitourinary system can bediagnosed by methods comprising determining from a sample derived from asubject an abnormally decreased or increased level of the IGS4polypeptide or IGS4 mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as an IGS4, in a sample derived from a host are well knownto those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

In another aspect, the present invention relates to a diagonostic kitfor a disease or suspectibility to a disease, particularly PNS,psychiatric and CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation, dyskinesias, Huntington's disease, Tourett's syndrome,tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders, which comprises:

-   -   (a) an IGS4 polynucleotide, preferably the nucleotide sequence        of SEQ ID NO: 1. SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or        a fragment thereof; and/or    -   (b) a nucleotide sequence complementary to that of (a); and/or    -   (c) an IGS4 polypeptide, preferably the polypeptide of SEQ ID        NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or of SEQ ID NO: 8, or a        fragment thereof; and/or    -   (d) an antibody to an IGS4 polypeptide, preferably to the        polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or of        SEQ ID NO: 8; and/or    -   (e) a RAMP polypeptide required for the relevant biological or        antigenic properties of an IGS4 polypeptide        It will be appreciated that in any such kit, (a), (b), (c) (d)        or (e) may comprise a substantial component. Preferably the        present invention relates to a diagnostic kit for diagnosing or        determining a disease or a susceptibility to a disease of the        nervous system, including the central nervous system (CNS) and        the peripheral nervous system (PNS), a disease of the        gastrointestinal system and/or of the cardiovascular system        and/or of skeletal muscle and/or of the thyroid, and/or also        lung diseases, immunological diseases and disorders of the        genitourinary system.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them if required together with relevant RAMP's, mayalso be used as immunogens to produce antibodies immunospecific for theIGS4 polypeptides. The term “immunospecific” means that the antibodieshave substantiall greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art.

Antibodies generated against the IGS4 polypeptides may be obtained byadministering the polypeptides or epitope-bearing fragments, analogs orcells to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique, which providesantibodies produced by continuous cell line cultures, may be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Naure (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against IGS4 polypeptides as such, or against IGS4polypeptide-RAMP complexes, may also be employed to treat PNS,psychiatric and CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation, dyskinesias, Huntington's disease, Tourett's syndrome,tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders, among others. Preferably the antibodies ofthe present invention may be used to treat disorders of the nervoussystem, including the central nervous system (CNS) and the peripheralnervous system (PNS), disorders of the gastrointestinal system and/or ofthe cardiovascular system and/or of skeletal muscle and/or of thethyroid, and/or also to treat lung diseases, immunological diseases anddisorders of the genitourinary system.

Animals

Another aspect of the invention relates to non-human animal-basedsystems which act as models for disorders arising from aberrantexpression or activity of IGS4. Non-human animal-based model systems mayalso be used to further characterize the activity of the IGS4 gene. Suchsystems may be utilized as part of screening strategies designed toidentify compounds which are capable to treat IGS4 based disorders suchas PNS, psychiatric and CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation, dyskinesias, Huntington's disease, Tourett's syndrome,tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer, diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders. In particular, the systems may be utilizedas part of screening strategies designed to identify compounds which arecapable in particular to treat IGS4 based disorders of the nervoussystem, including the central nervous system (CNS) and the peripheralnervous system (PNS), disorders of the gastrointestinal system and/or ofthe cardiovascular system and/or of skeletal muscle and/or of thethyroid, and/or also to treat lung diseases, immunological diseases anddisorders of the genitourinary system. In this way the animal-basedmodels may be used to identify pharmaceutical compounds, therapies andinterventions which may be effective in treating disorders of aberrantexpression or activity of IGS4. In addition such animal models may beused to determine the LD₅₀ and the ED₅₀ in animal subjects. These datamay be used to determine the in vivo efficacy of potential IGS4 disordertreatments.

Animal-based model systems of IGS4 based disorders, based on aberrantIGS4 expression or activity, may include both non-recombinant animals aswell as recombinantly engineered transgenic animals.

Animal models for IGS4 disorders may include, for example, geneticmodels. Animal models exhibiting IGS4 based disorder-like symptoms maybe engineered by utilizing, for example, IGS4 sequences such as thosedescribed, above, in conjunction with techniques for producingtransgenic animals that are well known to persons skilled in the art.For example, IGS4 sequences may be introduced into, and overexpressedand/or misexpressed in, the genome of the animal of interest, or, ifendogenous IGS4 sequences are present, they may either be overexpressed,misexpressed, or, alternatively, may be disrupted in order tounderexpress or inactivate IGS4 gene expression.

In order to overexpress or misexpress a IGS4 gene sequence, the codingportion of the IGS4 gene sequence may be ligated to a regulatorysequence which is capable of driving high level gene expression orexpression in a cell type in which the gene is not nornally expressed inthe animal type of interest. Such regulatory regions will be well knownto those skilled in the art, and may be utilized in the absence of undueexperimentation.

For underexpression of an endogenous IGS4 gene sequence, such a sequencemay be isolated and engineered such that when reintroduced into thegenome of the animal of interest, the endogenous IGS4 gene alleles willbe inactivated, or “knocked-out”. Preferably, the engineered IGS4 genesequence is introduced via gene targeting such that the endogenous IGS4sequence is disrupted upon integration of the engineered IGS4 genesequence into the animal's genome. Gene targeting is discussed, below,in this section.

Animals of any species, including, but not limited to, mice, rats,rabbits, squirrels, guinea-pigs, pigs, micro-pigs, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate animal models of IGS4 related disorders.

Any technique known in the art may be used to introduce a IGS4 transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to pronuclear microinjection(Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (van der Putten etal., Proc. Natl. Acad. Sci., USA 82:6148-6152, 1985); gene targeting inembryonic stem cells (Thompson et al., Cell 56:313-321, 1989,);electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1B14, 1983); andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723, 1989);etc. For a review of such techniques, see Gordon, Transgenic Animals,Intl. Rev. Cytol. 115:171-229, 1989, which is incorporated by referenceherein in its entirety.

The present invention provides for transgenic animals that carry theIGS4 transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. (See,for example, techniques described by Jakobovits, Curr. Biol. 4:761-763,1994) The transgene may be integrated as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal. (Lasko, M. et al., Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992).

The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art.

When it is desired that the IGS4 transgene be integrated into thechromosomal site of the endogenous IGS4 gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous IGS4gene of interest (e.g., nucleotide sequences of the mouse IGS4 gene) aredesigned for the purpose of integrating, via homologous recombinationwith chromosomal sequences, into and disrupting the function of, thenucleotide sequence of the endogenous IGS4 gene or gene allele. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene of interest in only thatcell type, by following, for example, the teaching of Gu et al. (Gu, H.et al., Science 265:103-106, 1994). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art.

Once transgenic animals have been generated, the expression of therecombinant IGS4 gene and protein may be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the IGS4 transgene in the tissues of the transgenicanimals may also be assessed using techniques which include but are notlimited to Northern blot analysis of tissue samples obtained from theanimal, in situ hybridization analysis, and RT-PCR. Samples of targetgene-expressing tissue, may also be evaluated immunocytochemically usingantibodies specific for the target gene transgene product of interest.The IGS4 transgenic animals that express IGS4 gene mRNA or IGS4transgene peptide (detected immunocytochemically, using antibodiesdirected against target gene product epitopes) at easily detectablelevels may then be further evaluated to identify those animals whichdisplay characteristic IGS4 based disorder symptoms.

Once IGS4 transgenic founder animals are produced (i.e., those animalswhich express IGS4 proteins in cells or tissues of interest, and which,preferably, exhibit symptoms of IGS4 based disorders), they may be bred,inbred, outbred, or crossbred to produce colonies of the particularanimal. Examples of such breeding strategies include but are not limitedto: outbreeding of founder animals with more than one integration sitein order to establish separate lines; inbreeding of separate lines inorder to produce compound IGS4 transgenics that express the IGS4transgene of interest at higher levels because of the effects ofadditive expression of each IGS4 transgene; crossing of heterozygoustransgenic animals to produce animals homozygous for a given integrationsite in order to both augment expression and eliminate the possible needfor screening of animals by DNA analysis; crossing of separatehomozygous lines to produce compound heterozygous or homozygous lines;breeding animals to different inbred genetic backgrounds so as toexamine effects of modifying alleles on expression of the IGS4 transgeneand the development of IGS4-like symptoms. One such approach is to crossthe IGS4 transgenic founder animals with a wild type strain to producean F1 generation that exhibits IGS4 related disorder-like symptoms, suchas those described above. The F1 generation may then be inbred in orderto develop a homozygous line, if it is found that homozygous target genetransgenic animals are viable.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises administering to (forexample by inoculation) the mammal the IGS4 polypeptide, or a fragmentthereof, if required together with a RAMP polypeptide, adequate toproduce antibody and/or T cell immune response to protect said animalfrom PNS, psychiatric and CNS disorders, including schizophrenia,episodic paroxysmal anxiety (EPA) disorders such as obsessive compulsivedisorder (OCD), post traumatic stress disorder (PTSD), phobia and panic,major depressive disorder, bipolar disorder, Parkinson's disease,general anxiety disorder, autism, delirium, multiple sclerosis,Alzheimer disease/dementia and other neurodegenerative diseases, severemental retardation, dyskinesias, Huntington's disease, Tourett'ssyndrome, tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders, among others. Yet another aspect of theinvention relates to a method of inducing immunological response in amammal which comprises delivering the IGS4 polypeptide via a vectordirecting expression of the IGS4 polynucleotide in vivo in order toinduce such an immunological response to produce antibody to protectsaid animal from diseases. In particular the invention relates to amethod for inducing an immunological response in a mammal whichcomprises inoculating the mammal with the IGS4 polypeptide, or afragment thereof, if required together with a RAMP polypeptide, adequateto produce antibody and/or T cell immune response to protect said animalfrom disorders of the nervous system, including the central nervoussystem (CNS) and the peripheral nervous system (PNS), disorders of thegastrointestinal system and/or of the cardiovascular system and/or ofskeletal muscle and/or of the thyroid, and/or also from lung diseases,immunological diseases and disorders of the genitourinary system.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to an IGS4 polypeptidewherein the composition comprises an IGS4 polypeptide or IGS4 gene. Suchimmunological/vaccine formulations (compositions) may be eithertherapeutic immunological/vaccine formulations or prophylacticimmunological/vaccine formulations. The vaccine formulation may furthercomprise a suitable carrier. Since the IGS4 polypeptide may be brokendown in the stomach, it is preferably administered parenterally(including subcutaneous, intramuscular, intravenous, intradermal etc.injection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

Screening Assays

The IGS4 polypeptide of the present invention may be employed in ascreening process for compounds which bind the receptor and whichactivate (agonists) or inhibit activation of (antagonists) the receptorpolypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands may be natural substrates and ligands or may be structural orfunctional mimetics.

IGS4 polypeptides are responsible for biological functions, includingpathologies. Accordingly, it is desirable to find compounds and drugswhich stimulate IGS4 on the one hand and which can inhibit the functionof IGS4 on the other hand. In general, agonists are employed fortherapeutic and prophylactic purposes for such conditions as PNS,psychiatric and CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation, dyskinesias, Huntington's disease, Tourett's syndrome,tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer; diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders. Antagonists may be employed for a varietyof therapeutic and prophylactic purposes for such conditions as PNS,psychiatric and CNS disorders, including schizophrenia, episodicparoxysmal anxiety (EPA) disorders such as obsessive compulsive disorder(OCD), post traumatic stress disorder (PTSD), phobia and panic, majordepressive disorder, bipolar disorder, Parkinson's disease, generalanxiety disorder, autism, delirium, multiple sclerosis, Alzheimerdisease/dementia and other neurodegenerative diseases, severe mentalretardation, dyskinesias, Huntington's disease, Tourett's syndrome,tics, tremor, dystonia, spasms, anorexia, bulimia, stroke,addiction/dependency/craving, sleep disorder, epilepsy, migraine;attention deficit/hyperactivity disorder (ADHD); cardiovasculardiseases, including heart failure, angina pectoris, arrhythmias,myocardial infarction, cardiac hypertrophy, hypotension,hypertension—e.g. essential hypertension, renal hypertension, orpulmonary hypertension, thrombosis, arteriosclerosis, cerebralvasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebralinfarction, peripheral vascular disease, Raynaud's disease, kidneydisease—e.g. renal failure; dyslipidemias; obesity; emesis;gastrointestinal disorders, including irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD),motility disorders and conditions of delayed gastric emptying, such aspost operative or diabetic gastroparesis, and diabetes, ulcers—e.g.gastric ulcer, diarrhoea; other diseases including osteoporosis;inflammations; infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; chemotherapy induced injury; tumor invasion; immune disorders;urinary retention; asthma; allergies; arthritis; benign prostatichypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus;and gynaecological disorders. Particularly, the present invention may beemployed in a screening process for compounds which bind the receptorand which activate (agonists) or inhibit activation of (antagonists) theIGS4 neuromedin receptor protein, preferably the mammalian IGS4neuromedin receptor protein, said protein exhibiting high affinitybinding for neuromedin U, preferably for neuromedin U-8, for neuromedinU-23 and/or for neuromedin U-25. These screening assays are particularlysuitable for screening compounds which are effective with regard todisorders of the nervous system, including the central nervous system(CNS) and the peripheral nervous system (PNS), disorders of thegastrointestinal system and/or of the cardiovascular system and/or ofskeletal muscle and/or of the thyroid, and/or also to lung diseases,immunological diseases and disorders of the genitourinary system.

In general, such screening procedures involve producing appropriatecells, which express the receptor polypeptide of the present inventionon the surface thereof and, if essential co-expression of RAMP's at thesurface thereof. Such cells include cells from mammals, yeast,Drosophila or E. coli. Cells expressing the receptor (or cell membranecontaining the expressed receptor) are then contacted with a testcompound to observe binding, or stimulation or inhibition of afunctional response.

One screening technique includes the use of cells which express thereceptor of this invention (for example, transfected CHO cells) in asystem which measures extracellular pH, intracellular pH, orintracellular calcium changes caused by receptor activation. In thistechnique, compounds may be contacted with cells expressing the receptorpolypeptide of the present invention. A second messenger response, e.g.,signal transduction, pH changes, or changes in calcium level, is thenmeasured to determine whether the potential compound activates orinhibits the receptor.

Another method involves screening for receptor inhibitors by determiningmodulation of a receptor-mediated signal, such as cAMP accumulationand/or adenylate cyclase activity. Such a method involves transfectingan eukaryotic cell with the receptor of this invention to express thereceptor on the cell surface. The cell is then exposed to an agonist tothe receptor of this invention in the presence of a potentialantagonist. If the potential antagonist binds the receptor, and thusinhibits receptor binding, the agonist-mediated signal will bemodulated.

Another method for detecting agonists or antagonists for the receptor ofthe present invention is the yeast-based technology as described in U.S.Pat. No. 5,482,835, incorporated by reference herein.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the receptor is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfaces.Inhibitors of activation are generally assayed in the presence of aknown agonist and the effect on activation by the agonist by thepresence of the candidate compound is observed.

Thus candidate compounds may be screened which show ligand binding tothe IGS4 receptors of the present invention. In the context of thepresent invention the term “ligand binding” is understood as to describecompounds with affinity to the IGS4 receptors showing log EC₅₀ values ofat least below −6.00 (approx. 660 nM), preferably log EC₅₀ below −7.00(approx. 55 nM), more preferably log EC₅₀ below −9.00 (approx. 500 pM to1.2 nM), and most preferably log EC₅₀ below −10.00 (approx. 50-100 pM).

Thus in one aspect the invention concerns a method of determiningwhether a substance is a potential ligand of IGS4 receptor comprising

-   -   (a) contacting cells expressing one of the IGS4 neuromedin        receptors defined supra or one of the receptors of SEQ ID NO:2,        SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8, or contacting a        receptor membrane preparation comprising one of said IGS4        neuromedin receptors defined supra or one of the receptors of        SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 with        labeled neuromedin U in the presence and in the absence of the        substance; and    -   (b) measuring the binding of neuromedin U to IGS4.

Further, the assays may simply comprise the steps of mixing a candidatecompound with a solution containing an IGS4 polypeptide to form amixture, measuring the IGS4 activity in the mixture, and comparing theIGS4 activity of the mixture to a standard.

The IGS4 cDNA, protein and antibodies to the protein may also be used toconfigure assays for detecting the effect of added compounds on theproduction of IGS4 mRNA and protein in cells. For example, an ELISA maybe constructed for measuring secreted or cell associated levels of IGS4protein using monoclonal and polyclonal antibodies by standard methodsknown in the art, and this can be used to discover agents which mayinhibit or enhance the production of IGS4 (also called antagonist oragonist, respectively) from suitably manipulated cells or tissues.Standard methods for conducting screening assays are well known in theart.

Examples of potential IGS4 antagonists include antibodies or, in somecases, oligonucleotides or proteins which are closely related to theligand of the IGS4, e.g., a fragment of the ligand, or small moleculeswhich bind to the receptor but do not elicit a response, so that theactivity of the receptor is prevented.

Thus in another aspect, the present invention relates to a screening kitfor identifying agonists, antagonists, ligands, receptors, substrates,enzymes, etc. for IGS4 polypeptides; or compounds which decrease,increase and/or otherwise enhance the production of IGS4 polypeptides,which comprises:

-   -   (a) an IGS4 polypeptide, preferably that of SEQ ID NO: 2, SEQ ID        NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8;    -   (b) a recombinant cell expressing an IGS4 polypeptide,        preferably that of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or        SEQ ID NO: 8;    -   (c) a cell membrane expressing an IGS4 polypeptide; preferably        that of SEQ ID NO: 2, SEQ ID NO: 4. SEQ ID NO: 6 or SEQ ID NO:        8; or    -   (d) antibody to an IGS4 polypeptide, preferably that of SEQ ID        NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.        It will be appreciated that in any such kit, (a), (b), (c)        or (d) may comprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions relatedto both an excess of and insufficient amounts of IGS4 activity.

If the activity of IGS4 is in excess, several approaches are available.One approach comprises administering to a subject an inhibitor compound(antagonist) as hereinabove described along with a pharmaceuticallyacceptable carrier in an amount effective to inhibit activation byblocking binding of ligands to the IGS4, or by inhibiting interactionwith a RAMP polypeptide or a second signal, and thereby alleviating theabnormal condition.

In another approach, soluble forms of IGS4 polypeptides still capable ofbinding the ligand in competition with endogenous IGS4 may beadministered. Typical embodiments of such competitors comprise fragmentsof the IGS4 polypeptide.

In still another approach, expression of the gene encoding endogenousIGS4 can be inhibited using expression-blocking techniques. Known suchtechniques involve the use of antisense sequences, either internallygenerated or separately administered. See, for example, O'Connor, JNeurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. USA (1988).Alternatively, oligonucleotides, which form triple helices with thegene, can be supplied. See, for example, Lee et al., Nucleic Acids Res(1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al,Science (1991) 251:1360. These oligomers can be administered per se orthe relevant oligomers can be expressed in vivo. Synthetic antisense ortriplex oligonucleotides may comprise modified bases or modifiedbackbones. Examples of the latter include methylphosphonate,phosphorothioate or peptide nucleic acid backbones. Such backbones areincorporated in the antisense or triplex oligonucleotide in order toprovide protection from degradation by nucleases and are well known inthe art. Antisense and triplex molecules synthesized with these or othermodified backbones also form part of the present invention.

In addition, expression of the IGS1 polypeptide may be prevented byusing ribozymes specific to the IGS1 mRNA sequence. Ribozymes arecatalytically active RNAs that can be natural or synthetic (see forexample Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33.)Synthetic ribozymes can be designed to specifically cleave IGS1 mRNAs atselected positions thereby preventing translation of the IGS1 mRNAs intofunctional polypeptide. Ribozymes may be synthesized with a naturalribose phosphate backbone and natural bases, as normally found in RNAmolecules. Alternatively the ribosymes may be synthesized withnon-natural backbones to provide protection from ribonucleasedegradation, for example, 2′-O-methyl RNA, and may contain modifiedbases.

For treating abnormal conditions related to an under-expression of IGS4and its activity, several approaches are also available. One approachcomprises administering to a subject a therapeutically effective amountof a compound which activates IGS4, i.e., an agonist as described above,in combination with a pharmaceutically acceptable carrier, to therebyalleviate the abnormal condition. Alternatively, gene therapy may beemployed to effect the endogenous production of IGS4 by the relevantcells in the subject. For example, a polynucleotide of the invention maybe engineered for expression in a replication defective retroviralvector, as discussed above. The retroviral expression construct may thenbe isolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For overview of gene therapy, seeChapter 20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics,Strachan T. and Read A. P., BIOS Scientific Publishers Ltd (1996).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Formulation and Administration

Peptides, such as the soluble form of IGS4 polypeptides, and agonistsand antagonist peptides or small molecules, may be formulated incombination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible.

The dosage range required depends on the choice of peptide or compound,the route of administration, the nature of the formulation, the natureof the subject's condition, and the judgment of the attendingpractitioner. Suitable dosages are in the range of 0.1-100 μg/kg ofsubject. Wide variations in the needed dosage, however, are to beexpected in view of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

The following examples are only intended to further illustrate theinvention in more detail, and therefore these examples are not deemed torestrict the scope of the invention in any way.

EXAMPLE 1 The Cloning of CDNA Encoding a Novel G Protein-CoupledReceptor Example 1a Homology PCR Cloning of a Genomic Fragment Encodinga Novel G-Protein Coupled Receptor (GPCR)

A PCR based homology cloning strategy was used to isolate partialgenomic DNA sequences encoding novel G-protein coupled receptors.Forward (F22) and reverse (R44 and R46) degenerate PCR primers weredesigned in conserved areas of the neurotensin receptor gene family(Vita N. et al. [1993] Febs Lett. 317, 139-142; Vita N. et al. [1998]Eur. J. Pharmacol. 360, 265-272) within transmembrane domains 1 (TM1)and 3 (TM3) and at the boundary between TM3 and intracellular loop 2(12):

F22 (TM1): (SEQ ID NO: 13) 5′-CTCATCTTCGCGGTGGGC(A or G)C(A,C,G or T)G(C or T)(A,C,G or T)GG-3′ R44 (TM3/I2): (SEQ ID NO: 14)5′-GGCCAGGCAGCGCTCCGCGCT(C or Inosine)A(A or G) (A,C,G or T)C(C orT)(A,C,G or T)GC(A,G or T)-3′ R46 (TM3): (SEQ ID NO: 15) 5′-GAA(A orG)TA(A or G)TAGCC(A or G)CG(A or G) CAGCC(A or T)-3′

In order to suppress amplification of known members of the neurotensinreceptor family, the 3′ ultimate nucleotide position of primer R44 waschosen in such a way that is was not complementary to the correspondingposition of both NTR1 and NTR2 cDNA. The primary PCR reaction wascarried out in a 60 μl volume and contained 100 ng human genomic DNA(Clontech), 6 μl GeneAmp™ 10×PCR buffer II (100 mM Tris-HCl pH 8.3; 500mM KCl, Perkin Elmer), 3.6 μl 25 mM MgCl₂, 0.36 μl dNTPs (25 mM of eachdNTP), 1.5 units Ampliaq-Gold™ polymerase (Perkin Elmer) and 30 pmolesof each of the degenerated forward (F22) and reverse primer (R44).Reaction tubes were heated at 95° C. for 10 min and then subjected to 35cycles of denaturation (95° C., 1 min), annealing (55° C., 2 min) andextension (72° C., 3 min). Finally reaction tubes were heated for 10 minat 72° C.

For the semi-nested PCR reaction 1 μl of a 1/50 dilution of the primaryPCR reaction was used as a template using the degenerate forward andreverse primers F22 and R46 respectively. The semi-nested PCR reactionwas carried out under the same conditions as the primary PCR reaction.

Semi-nested PCR reaction products were size fractionated on an agarosegel and stained with ethidium bromide. Although a fragment of ±220 bpwas expected, only a fragment of ±120 bp was visible. This fragment waspurified from gel using the Qiaex-II™ purification kit (Qiagen) andligated into the pGEM-T plasmid according to the procedure recommendedby the supplier (pGEM-T kit, Promega). The recombinant plasmids thusproduced were used to transform competent E. coli SURE™ 2 bacteria(Stratagene). Transformed cells were plated on LB agar plates containingampicillin (100 μg/ml), IPTG (0.5 mM) and X-gal (50 μg/ml). Plasmid DNAwas purified from mini-cultures of individual colonies using aQiagen-tip 20 miniprep kit (Qiagen). DNA sequencing reactions werecarried out on the purified plasmid DNA with the ABI Prism™ BigDye™Terminator Cycle Sequencing Ready Reaction kit (PE-ABI), usinginsert-flanking primers.

Table 7 Overview of oligo primers used. SEQ ID NO: 13 F22:5′-CTCATCTTCGCGGTGGGC(A or G)C (A,C,G or T)G(C or T)(A,C,G or T)GG- 3′SEQ ID NO: 14 R44: 5′-GGCCAGGCAGCGCTCCGCGCT(C or Inosine)A(A or G)(A,C,Gor T)C(C or T)(A,C,G or T)GC(A,G or T)-3′ SEQ ID NO: 15 R46: 5′-GAA(A orG)TA(A or G)TAGCC(A or G)CG(A or G)CAGCC(A or T)-3′ SEQ ID NO: 16 AP1:5′-CCATCCTAATACGACTCACTATAGGGC- 3′ SEQ ID NO: 17 AP2:5′-ACTCACTATAGGGCTCGAGCGGC-3′ SEQ ID NO: 18 IGS4R1:5′-GGATCCCAAATAAGAAAGGGTAGTT GC-3′ SEQ ID NO: 19 IG54R2:5′-AAAGGGTAGTTGCGCCACATCTCAT AGAC-3′ SEQ ID NO: 20 IGS4F5:5′-AGGTCTATGAGATGTGGCGCAACTA CCCT-3′ SEQ ID NO: 21 IGS4F6:5′-ATGTGGCGCAACTACCCTTTCTTAT TTGGG-3′ SEQ ID NO: 22 R74:5′-CGGAAGTTGGCGGACACG(A or G)(A, C or G)(A or G)TT(A or G)TA-3′ SEQ IDNO: 23 IG54F7: 5-GCTCAGCTTGAAACAGAGCCTCGTAC C-3′ SEQ ID NO: 24 IG54F8:5-CCATGTGGATCTACAATTTCATCATC C-3′ SEQ ID NO: 25 IGS4F9:5′-AAGACAAATCTCTTGAGGCAGATGA AGGG-3′ SEQ ID NO: 26 IGS4F10:5′-GATGCTGTTTGTCTTGGTCTTAGT GTTTGC-3′ SEQ ID NO: 27 IGS4R5:5′-GGATGATGAAATTGTAGATCCACAT GGGC-3′ SEQ ID NO: 28 IGS4R6:5′-TGTGGAGAAGTCTCTCAAAGTGTG G-3′ SEQ ID NO: 29 IGS4R7:5-TAGTAGGAGTGACAGCCTGACTCGGA ACG-3′ SEQ ID NO: 30 IGS4R8:5-AACGTAGATGACTCAGGACGAACCAT TTCC-3′ SEQ ID NO: 31 IGS4F11:5-TCGTACCAGGGGAGGCTCAGGC-3′Sequencing reaction products were purified via EtOH/NaOAc precipitationand analyzed on an ABI 377 automated sequencer.Sequence analysis of the insert of clone HNT1552 showed that itpotentially encoded part of a novel member of the GPCR family. We referto this novel GPCR sequence as IGS4.

Example 1b Cloning of cDNA Fragments Containing the Complete IGS4 CodingSequence

The complete coding sequence of IGS4 cDNA was obtained via both RACEanalysis (rapid amplification of cDNA ends) and RT-PCR amplification.5′- and 3′ RACE PCR reactions were performed on Marathon-Ready™ cDNAfrom human brain or testis (Clontech n° 7400-1 and 7414-1 respectively),using the adaptor primers 1 and 2 (API: SEQ ID NO: 16 ; AP2: SEQ ID NO:17) provided with the Marathon™ cDNA amplification kit (ClontechK1802-1) and IGS4 specific primers. PCR RACE reactions were performedaccording to the instructions of the Marathon-Ready™ cDNA user manualprovided by Clontech. RACE products were separated on agarose gel,visualized with ethidium bromide and blotted onto Hybond N⁺ membranes.Blots were prehybridized at 65° C. for 2 h in modified Church buffer(0.5M phosphate, 7% SDS, 10 mM EDTA) and then hybridised overnight at65° C. in the same buffer containing 2×10⁶ cpm/ml of a ³²P-labelled IGS4cDNA probe. IGS4 cDNA probes were radiolabelled via random primedincorporation of [α-³²P]dCTP to a specific activity of >10⁹ cpm/μg usingthe Prime-It II kit™ (Stratagene) according to the instructions providedby the supplier. Hybridized blots were washed at high stringency (2×30min at room temperature in 2×SSC/0.1% SDS, followed by 2 washes of 40min at 65° C. in 0.1×SSC, 0.1% SDS) and autoradiographed overnight.Hybridizing fragments were purified from a preparative gel, cloned intothe pGEM-T vector and sequenced as described above.

An initial round of semi-nested 5′ RACE analysis on human brain cDNAusing the IGS4 specific primers IGS4R1 (SEQ ID NO: 18) and IGS4R2 (SEQID NO: 19) (designed on the DNA sequence of clone HNT1552) yieldedclones HNT1886 and HNT1887 (FIG. 1). These clones extended the IGS4 cDNAsequence upstream up to and beyond the putative start of translationcodon. Likewise an initial round of 3′ RACE analysis on human brain cDNAusing IGS4 specific primers IGS4F5 (SEQ ID NO: 20) and IGS4F6 (SEQ IDNO: 21) yielded clones HNT1874-1878 and HNT1902-1903 (FIG. 1). Theseclones extended the known IGS4 cDNA at the 3′ end.

All sequences obtained at this point were assembled into a single contigwhich contained a long open reading frame, encoding part of a novelprotein that was most similar to human orphan receptor FM-3 (Tan et al.,Genomics 52, 223-229 [1998], GenBank accession n° AF044600 andAF044601). To investigate the RNA expression profile of IGS4, a MasterBlot™ membrane (Clontech cat n° 7770-1) containing RNA from differenthuman tissues was hybridized to the ³²P-labelled insert of clone HNT1903under the conditions recommended by the supplier. The strongesthybridization was obtained with testis RNA whereas much weaker signalswere obtained in prostate, stomach, spinal cord, hippocampus, medullaoblongata, thyroid gland, thymus, lung and trachea.

Since the contig sequence did not yet contain the complete IGS4 codingsequence we set up an RT-PCR homology cloning experiment on human totalbrain RNA using IGS4 specific primer IGS4F6 (SEQ ID NO: 21) and adegenerated primer (R74, SEQ ID NO: 22), which was designed in aconserved area (at the TM7/C-terminal intracellular part) of the GPCRsubfamily composed of the neurotensin receptors 1 and 2, the growthhormone secretagogue receptor (Howard A. D. et al. [1996] Science 273,974-977) and the orphan GPCR FM-3 and GPR38 (McKee K. K. et al. [1997]Genomics 46, 426-434). RT-PCR reactions were carried out in a 50 μlvolume on 500 ng total RNA from human brain using the Titan™ One TubeRT-PCR System (Boehringer catalogue n° 1,888,382) according to therecommendations of the supplier. Briefly, RT-PCR conditions were asfollows: reverse transcription for 45 min at 55° C.; 2 min denaturationat 94° C., followed by a touch-down PCR reaction of 20 cycles (30 secdenaturation at 94° C., 30 sec annealing at 60° C. [−0.25° C./cycle] and2 min extension at 68° C.) and an additional round of 30 PCR cycles (30sec denaturation at 94° C., 30 sec annealing at 55° C. and 3 min [+5sec/cycle] extension at 68° C.). This was concluded with an extraextension step of 7 min at 68° C. Reaction products were analyzed viaSouthern blotting using the radiolabelled insert of clone HNT1903. Afragment of ±690 bp that hybridized to the probe was purified from thegel (Qiaexil™, Qiagen) and cloned into the pGEM-T vector yielding clonesHNT2210-2212. Sequence analysis of these clones allowed to extend theexisting IGS4 cDNA contig in the 3′ direction.

Since the extended IGS4 cDNA contig still did not yet contain atranslational stop codon, additional IGS4 specific 3′ RACE primers weredesigned (IGS4F7-10, SEQ ID NO: 23-26)). Nested or semi-nested 3′ RACEreactions were carried out on Marathon Ready™ cDNA from human testis(Clontech 7414-1). IGS4 specific bands (as assessed via Southern blotanalysis using an IGS4 specific probe) were cloned into pGEM-T. Thisyielded clones HNT2289-90 (AP1/IGS4F5->AP2/IGS4F9), HNT2293-2295(AP1/IGS4F6->AP2/IGS4F9), HNT2296-2297 (AP1/IGS4F7->AP2/IGS4F9),HNT2308-2310 (AP1/IGS4F8->AP2/IGS4F10) HNT2253 (AP1/IGS4F7->AP1/IGS4F5).An additional 5′ RACE PCR reaction carried out on testis Marathon Ready™cDNA yielded clones HNT2279-2281 (AP2/IGS4R6->AP2/IGS4R5). (note:AP1/IGS4F5->AP2/IGS4F9 e.g. indicates that clones were generated from anIGS4 specific fragment obtained after the primary RACE PCR reaction[using primer pair AP1/IGS4F5] was nested with primer pair AP2/IGS4F9).

Sequence analysis of these clones allowed to extend the existing IGS4cDNA contig further in the 3′ direction although the end of the IGS4coding sequence was not yet been reached. A computer-assisted homologysearch (Blastn; Altschul S. F. et al., Nucleic Acids Res. (1997)25:3389-3402) of the IGS4 contig DNA sequence against the expressedsequence tag (EST) database (dbest) showed the presence of an ESTsequence (accession n° N45474) which overlapped with the 3′ end of theIGS4 contig (near 100% identity in the overlap area). EST N45474 furtherextended the IGS4 DNA contig at the 3′ end into a translational stopcodon and into the 3′ untranslated region (3′-UTR). In addition anotherset of ESTs was identified which all covered the 3′-UTR of the IGS4 mRNA(FIG. 2). Additional IGS4 specific primers (IGS4R7-8, SEQ ID NO: 29-30))were designed within the 3′-UTR of these ESTs. Primary PCR reactionswere carried out on Marathon Ready™ cDNA from human testis using variouscombinations of the IGS4F7 (SEQ ID NO: 23), IGS4F1 I (SEQ ID NO: 31) andIGS4R7-8 (SEQ ID NO: 29-30) primers. PCR tubes were heated for 2 min at95° C. and then subjected to 35 cycles of denaturation (95° C., 30 sec),annealing (65° C., 30 sec) and extension (72° C., 1 min 30 sec). Finallythe reactions tubes were heated at 72° C. for 10 min. Nested PCRreactions were also carried out under the same conditions. DNA fragmentsof ±1630 bp were purified from gel and cloned into the pGEM-T vector.The following clones were obtained: HNT2311, HNT2312 and HNT2317(IGS4F7/IGS4R7->IGS4F11/IGS4R8); HNT2313, HNT2324, HNT2326 and HNT2328(IGS4F11->IGS4R8); HNT2314, HNT2315 and HNT2322 (IGS4F11->R7). CloneHNT2363 was obtained from a purified 1630 bp PCR fragment, that wasamplified from human testis Marathon Ready™ cDNA using the IGS4F 11/R7primer pair under the following slightly modified conditions. After aninitial denaturation of 2 min at 94° C., PCR tubes were subjected to 15cycles of denaturation [15 sec, 94° C.], annealing [30 sec, 65° C.] andextension [2 min, 72° C.] followed by another 20 cycles of denaturation[15 sec, 94° C.], annealing [30 sec, 65° C.] and extension [2 min, 72°C.; +10 sec/cycle]. There was a final extension step of 7 min at 72° C.Sequence analysis of these clones allowed to assemble an IGS4 cDNAconsensus sequence (FIG. 1). Close inspection of all clones showed thatthey actually were of 2 sequence types, which differed at 5 nucleotidepositions. These variant sequences correspond to a polymorphism withinthe human population. We refer to these different cDNA types as IGS4ADNA(SEQ ID NO: 1 and SEQ ID NO: 3) and IGS4BDNA (SEQ ID NO: 5 and SEQ IDNO: 7). The consensus sequence contained a long open reading frame thatcontained two in-frame start codons (positions 55-57 (SEQ ID NO: 1 andSEQ ID NO: 5) and 64-66 (SEQ ID NO: 3 and SEQ ID NO: 7) in IGS4ADNA andIGS4BDNA), predicting a protein of either 415 (SEQ ID NO: 2 and SEQ IDNO: 6) or 412 (SEQ ID NO: 4 and SEQ ID NO: 8) amino acids, which showedgood homology to GPCR proteins. Hydropathy analysis (Kyte J. etal.[1982] J. Mol. Biol. 157: 105-132; Klein P. et al.[1985] Biochim.Biophys. Acta 815:468-476) of the protein also indicated the presence of7 transmembrane domains. Since the first ATG initiator codon is within aweak “Kozak” translation initiation context and the second one is in astrong Kozak context, it is likely that the IGS4A/B protein starts atthe second methionine and is 412 amino acids long (Kozak M. [1999] Gene234, 187-208). However some (or even exlusive) initiation at the firstATG cannot be excluded. Among the five polymorphic nucleotides, four(positions 947, 999, 1202 and 1216 in IGS4A/BDNA) resulted in a switchin the encoded amino acid residue, whereas the fifth (pos 1381 inIGS4A/BDNA) was within the 3′-UTR. The respective predicted proteinsequences are referred to as IGS4APROT (SEQ ID NO: 2 and SEQ ID NO: 4)and IGS4BPROT (SEQ ID NO: 6 and SEQ ID NO: 8). (note 1: the sequence ofIGS4APROT and IGS4BPROT in this document is represented as the longestpossible (415 amino acids) sequence but it is understood that the actualprotein might be 3 amino acids shorter at the amino-terminus; for thisreason the first 3 amino acids of IGS4APROT and IGS4BPROT in Table 4 and5 have been bracketed) (note 2: In this document IGS4 refers to the IGS4sequence in general, irrespective of the particular allelic type).Homology searches of the IGS4 protein sequence against public domainprotein databanks showed best homology to the human orphan GPCR FM-3(accession no 043664, Tan C. P., et al. Genomics (1998) 52: 223-229; 46%identity in IGS4A amino acid residues 26-342).

Homology searches of DNA databanks with the IGS4 cDNA sequence yielded anumber of entries which were also derived from the IGS4 gene locus (FIG.2 for overview):

-   -   10 EST sequence entries (accession nrs W61169, AI432384, W61131,        AI023570, F01358, F03770, Z38158, R40869, R37725, H11333), 2 STS        (sequence tagged sites) (accession nrs G20615 and G05725) and        one genomic sequence (accession nr AQ078563) were discovered        which were all derived from the 3′-UTR of IGS4 cDNA.    -   EST accession n° N45474 encoded the 3′ end of the IGS4 coding        sequence and part of the 3′ UTR (cfr supra).    -   A ‘working draft’ high throughput genomic sequence (accession nr        AC008571, version AC008571.1, deposited 3 AUG 1999), which        consisted of 42 unordered contigs assembled in an arbitrary        order was discovered in which we detected the entire IGS4 cDNA        sequence in 4 separate areas. These areas most likely correspond        to the different IGS4 exons as they were flanked by canonical        splice donor and acceptor sequences. On the basis of this        analysis the position of the different exons in the IGS4ADNA (or        IGS4BDNA) sequence can be defined as follows: exon1 (1-780),        exon 2 (781-865), exon 3 (866-991) and exon 4 (992-1658). The        AC008571 genomic sequence is of the IGS4A allelic type.    -   6 overlapping EST entries (accession nrs H11359, R13890, R13353,        F07531, F05108, F05107) were discovered of which the assembled        DNA sequence overlapped at its 3′ end with IGS4 exon2 and the        beginning of exon 3. However the DNA sequence upstream of exon 2        was completely different from IGS4 exon1. Probably these six        ESTs are derived from transcripts which originated from an        alternative promoter.    -   Finally 2 genomic sequence entries (accession nrs AQ019411 and        AQ015065) were discovered which encoded exon 2.

Among the many IGS4 cDNA clones that we isolated in the differentexperiments described above, we also discovered a number of clones thatcontained a 64 bp deletion (pos 866-929 in IGS4ADNA) (besides a numberof clones derived from unspliced [or partially spliced] transcripts). Sofar we only discovered truncated transcripts of the polymorphic type A.We refer to this splice variant cDNA sequence as IGS4A-64DNA (SEQ ID NO:9 and SEQ ID NO: 11). Since this deletion occurs exactly at the exon2/exon 3 boundary and since the last 2 nucleotides of the deletedfragment are “AG”, it is likely that this deletion represents analternative splicing event in which the “AG” within exon 3 served as asplice acceptor. The IGS4A reading frame encoded by the splice variantis frameshifted beyond the deletion point. The encoded (truncated)protein of 296 amino acids is referred to as IGS4A-64PROT (SEQ ID NO: 10and SEQ ID NO: 12). Hydropathy analysis of the IGS4A-64PROT sequenceshows that this protein only contains 5 transmembrane domains(corresponding to TM domains 1-5 of IGS4APROT). This truncated receptormight have physiological relevance.

The bacterial strain harboring plasmid HNT2322 (containing the IGS4ADNAinsert) was recloned after replating on LB agar plates containing 100 μgampicillin/ml and deposited both in the Innogenetics N.V. strain list(ICCG4320) and at the “Centraalbureau voor Schimmelculturen (CBS)” inBaarn, The Netherlands (accession n° CBS102221). Plasmid DNA wasprepared from the recloned isolate and the insert was resequenced andfound to be identical to the IGS4ADNA sequence.

The bacterial strain harboring plasmid HNT2363 (containing the IGS4BDNAinsert) was recloned after replating on LB agar plates containing 100 μgampicillin/ml and deposited both in the Innogenetics N.V. strain list(ICCG4340) and at the “Centraalbureau voor Schimmelculturen (CBS)” inBaarn, The Netherlands (accession n° CBS102222). Plasmid DNA wasprepared from the recloned isolate and the insert was resequenced andfound to be identical to the IGS4BDNA sequence.

EXAMPLE 2 Specific Changes in Intracellular Calcium ConcentrationsInduced in Chogα16-IGS4 Cells by Neuromedin U Example 2a ExperimentalProcedures: Method and Materials

A. Method and Materials for IGS-4 transfected CHOGα16-IGS4 cells.

The following materials were used in the experiments: Vector containingIGS4-DNA sequence (IGS4-pcDNA3.1); SuperFect Transfection Reagent(Qiagen); Nut-Mix F12 (Gibco) with 10% ECS, 0.028 mg/ml Gentamycin(Gibco); 0.22 mg/ml Hygromycin (Gibco).

Materials used for clone selection: Nut-Mix F12 with 10% FCS; 0.028mg/ml Genatmycin; 0.22 mg/ml Hygromycin and 0.55 mg/ml Geneticin(Gibco).

The following method was applied: Transfection with SuperFectTransfection Reagent was carried out as described by the manufacturer(Qiagen). Cells were plated in 24-well plates to 50% confluence. Perwell 0.6 μg/μl plasmid-DNA with 1 μl SuperFect Transfection Reagent wasadded. After 24 hours the medium was changed and transfected cell cloneswere selected by Geneticin-containing selection-medium. IGS4 expressingcell clones were characterized by RT-PCR and Northern Blot.

B. Method and Materials for FLIPR-Assay.

Cell Preparation:

For cell preparation the following materials were employed: plates:clear, flat-bottom, black well 96-well plates (Costar); Media: growthmedium: Nut-Mix F-12 (HAM) with Glutamax (Gibco) supplemented with 10%fetal calf serum (Gibco); Incubator: 5% CO₂, 37° C. (Nuaire).

The method was performed as follows: Cells were seeded 24 hours or 48hours prior to the experiment into black wall microplates. The celldensity was 0.8×10⁴ cells/well for 48 hour incubation and 2.2×10⁻⁴cells/well for 24 hour incubation. All steps were done under sterileconditions.

Dye Loading:

In order to observe changes in intracellular calcium levels, cells mustbe ‘loaded’ with a calcium-sensitive fluorescent dye. This dye, calledFLUO-4 (Molecular Probes) is excited at 488 nm, and emits light in the500-560nm range, only if a complex with calcium is formed. The dye wasused at 4 μM final concentration. Pluronic acid was added to increasedye solubility and dye uptake into the cells. Probenicid, an anionexchange protein inhibitor, was added to the dye medium to increase dyeretention in the cells.

The following materials were used:

-   -   2 mM dye stock: 1 mg Fluo-4 (Molecular Probes) solubilized in        443 μl low-water DMSO (Sigma). Aliquots stored at −20.    -   20% pluronic acid solution: 400 mg pluronic acid (Sigma)        solubilized in 2 ml low-water DMSO (Sigma) at 37° C. Stored at        room temperature.    -   Dye/pluronic acid mixture: Immediately before use, equal volumes        of the dye stock and 20% pluronic acid were mixed. The dye and        pluronic acid had a final concentration of 1 mM and 10%,        respectively.    -   Probenicid. 250 mM stock solution: 710 mg probenicid (Sigma)        solubilized in 5 ml 1N NaOH and mixed with 5 ml Hank's BSS        without phenol red (Gibco) supplemented with 20 mM HEPES.    -   Loading-Buffer: 10.5 ml Hank's BSS without phenol red (Gibco)        supplemented with 20 mM HEPES, 105 μl probenicid, 210 μl 1M        HEPES.    -   Wash-Buffer: Hank's BSS without phenol red (Gibco) supplemented        with 20 mM HEPES (Gibco) and 2.5 mM probenicid.

The method was worked as follows: The 2 mM stock of dye was mixed withan equal volume of 20% (w/v) pluronic acid immediately before adding tothe loading-Buffer. The growth-medium was aspirated out of the wellwithout disturbing the confluent cell layer. 100 l loading medium wasdispensed into each well using a Multidrop (Labsystems). Cell wereincubated in a 5% CO₂, 37° C. incubator for 30 minutes. In order tocalculate the background fluorescence, some wells were not dye loaded.The background fluorescence in these wells results from autofluorescenceof the cells. After dye loading, cell were washed three times withWash-Buffer (automated Denley cell washer) to reduce the basalfluorescence to 20.000-25.000 counts above background. 100 l buffer wasadded and cell were incubated at 37° C. till start of the experiment.

C. Preparation of Compound Plates.

The peptides were prepared at 3 μM (3× the final concentration) forinitial screening. For concentration response curves peptide-solutionswere prepared in concentration ranges from 30 μM to 100 nM. All peptideswere diluted in buffer containing 0.1% BSA (Sigma).

The following materials were used: Peptides: porcine Neuromedin U25, ratNeuromedin U-23, porcine Neuromedin U-8 (Bachem); Dilutionbuffer: Hank'sBSS without phenol red (Gibco) supplemented with 20 mM HEPES (Gibco) and0.1% BSA (Sigma); plates: clear, flat-bottom, 96-well plates (Costar).

D. Assay.

The FLIPR setup parameters were set to 0.4 sec exposure length, filter1, 50 μl fluid addition, pipettor height at 125 μl, Dispense Speed 40μl/sec without mixing.

Example 2b Results

To identify the endogenous ligand for the orphan G protein coupledreceptor (GPCR) IGS4, IGS4 (both forms iGS4A and IGS4B) was stablytransfected in Chinese Hamster Ovary (CHO) cells. Since the G proteincoupling mechanism of IGS4 was unknown, a specific CHO-cell strain wasused. These CHO-cells stably express the G-protein Gα16 (CHOGα16,Molecular Devices), which is known as “universal adapter” for GPCRs(Milligan G., Marshall F. and Rees S. (1996), Gα16 as a universal Gprotein adapter:.implications for agonist screening strategies. TIPS17:235-237).

The resulting CHOGα16-IGS4 cells were functionally screened on aFluorometric Imaging Plate Reader (FLIPR) to measure mobilisation ofintracellular calcium in response to putative ligands. At theconcentration of 10 nM, neuromedin U-23 induced a large, transient androbust calcium-response. In contrast, CHOGα16 cells and CHOGα16 cellsexpressing another, unrelated orphan GPCR, did not respond to neuromedinU-23. The results of these experiments with IGS4B are shown in FIG. 4.

Furthermore, the concentration dependence of IGS4 activation by porcineand rat neuromedin U isoforms were investigated (for both forms IGS4Aand IGS4B). In the range of 10⁻⁶-10⁻¹² M porcine neuromedin U-25, ratneuromedin U-23, porcine neuromedin U-8 induced specific IGS4-mediatedcalcium mobilisation in the FLIPR assay. All three Neuromedin U isoformstested caused the same maximal activation of IGS4B with LogE.C₅₀ valuesof −10.09±0.08 (neuromedin U-8, n=4; 80 pM), −10.61.±0.08 (neuromedinU-23, n=10; 50 pm) and −9.14±0.09 (neuromedin U-25, n=3; 1.2 nM). Thus,all three peptides cause potent activation of in particular IGS4B,suggesting that neuromedin U is the natural agonist for this receptor.The results of these experiments with IGS4B are shown in FIG. 3 a(neuromedin U-8), FIG. 3 b (neuromedin U-23) and FIG. 3 c (neuromedinU-25).

For the IGS4A receptor somewhat lower affinities were found, but stillshowing that the neuromedin U peptides are good ligands for IGS4receptors in general. The log EC₅₀ values found for IGS4A were asfollows; for neuromedin U-8: log EC₅₀=−9.3±0.09 (n=1; 485 pM); forneuromedin U-23: log EC₅₀=−7.27±0.16 (n=6; 53 nM); and for neuromedinU-25: log EC₅₀=−6.18±0.14 (n=3; 658 nM).

The calcium mobilisation response seen following activation of IGS4 byneuromedin U suggests that this receptor is coupled to G proteins of theGq/11 subfamily. In addition, basal levels of intracellular cAMP werenot modulated by porcine neuromedin U-8 (1 and 10 μM) in CHOGα16-IGS4cells, suggesting that this receptor does not couple to G proteins ofthe Gs subfamilies (data not shown).

EXAMPLE 3 IGS4 Hybridization on Human Multiple Tissue Expression Array(MTE™)

Human IGS4A DNA (±730 bp BamHI/HindIII insert from pGEMT-hIGS4A [ICCG#4320]) was radiolabelled via random primed incorporation of [^(s)_(x)−³²P]-dCTP to a specific activity of >10⁹ cpm/μg using the Prime-ItII kit™ (Stratagene). The labeled probe was purified from free label viaSephadex G-50 chromatography, denatured for 5 min. at 95° C. and addedto the ExpressHyb hybridization solution at a final concentration of1-1.5×10⁶ cpm/ml. The human Multiple Tissue Expression (MTE™) Array(Clontech # 7775-1) was prehybridized and hybridized in ExpressHybsolution at 65° C. for 30 min and overnight respectively according tothe recommendations of the supplier.

The hybridized MTE™ array was washed 5 times for 20 min in 2×SSC, 1% SDSat 65° C. and then 2 times for 20 min at 55° C. in 0.1×SSC, 0.5% SDS.After the washes the array was autoradiographed via phosphorimaging(Cyclone Storage Phosphor System, Packard) (FIG. 5). Hybridization dataof the MTE™ array were analyzed quantitatively using the OptiQuant ImageAnalysis Software (Packard). Signal intensity of different spotpositions containing RNA was corrected for the average background signalobtained from empty positions. The signal intensity obtained from thespot containing E. coli DNA was considered to represent a sampleexhibiting no IGS4 expression. Samples with signal intensities belowthat of E. coli DNA were considered to be negative.

Hybridization signals for different tissues on the RNA array have beenrecalculated by subtracting each value with the hybridization signalobserved for E. coli DNA (which is considered as the background signal).All tissues showing a lower hybridization signal are considered to bebelow background and to be IGS4 negative. Expression levels relative tothat found in testis (100%) have been plotted and are provided in FIG.7.

EXAMPLE 4 Tissue Distribution of IGS4 by Northern Blot Analysis

Human IGS4A DNA (±730 bp BamHI/HindIII insert from pGEMT-hIGS4A [ICCG#4320]) was radiolabelled via random primed incorporation of [^(s)_(x)−³²P]-dCTP to a specific activity of >10⁹ cpm/μg using the Prime-ItII kit™ (Stratagene). The labeled probe was purified from free label viaSephadex G-50 chromatography, denatured for 5 min. at 95° C. and addedto the ExpressHyb hybridization solution at a final concentration of1-1.5×10⁶ cpm/ml. The human Northern blots (Clontech #7760-1, #7759-1,#7767-1, #7755-1 and #7769-1) were prehybridized and hybridized inExpressHyb solution at 65° C. for 30 min and overnight respectivelyaccording to the recommendations of the supplier.

After hybridization Northern blots were washed 4 times 10 min at roomtemperature in 2×SSC, 0.05% SDS and then 2 times 40 min at 50° C. in0.1×SSC, 0.1% SDS. After the washes the Northern blots wereautoradiographed using phosphor storage plates (Cyclone Storage PhosphorSystem, Packard) and X-ray films. Results of Northern blots are shown inFIG. 6.

The results of the Northern blot analysis appear to be largelyconsistent with those from the array hybridization (Example 3). Thestrongest signal (2.4 kb transcript) by far is found in testis. A weak2.4 kb band was found in thymus, spinal cord, medulla, thyroid,thalamus, substantia nigra and a very weak band in corpus callosum,caudate. nucleus and stomach. For some tissues no 2.4 kb band could beseen on Northern whereas a strong to moderate hybridization signal wasobserved on the MTE array (e.g. whole brain, cerebral cortex, lung,temporal and frontal lobe, amygdala, cerebellum, kidney andhippocampus).

EXAMPLE 5 Quantitative RT-PCR Analysis

IGS4 expression levels in different human tissues were also determinedvia real-time quantitative RT-5 PCR (Q-PCR) using the LightCycler™instrument (Roche Diagnostics) and IGS4 specific TaqMan™ probes.

Example 5a Experimental Procedures

cDNA synthesis.

Prior to reverse transcription 3 μg total RNA from the human total RNApanels I to V (Clontech # K4000-1 to K4004-1) was treated with 3U DNAseI (Life Technologies # 18068-015) in a 30 μl reaction volume (20 mM TrispH 8.3, 50 mM KCl, 2 mM KCl) for 15 min at room temperature to destroypossibly contaminating genomic DNA. The reaction was stopped by adding 3μl 25 mM EDTA and heating for 10 min at 65° C. 2,6 μg of the DNAsetreated RNA was annealed with 1,3 μg oligo(dT) (Life Technologies #18418-012) and subjected to reverse transcription using the Omniscriptreverse transcriptase (Qiagen cat n° 205111) for 1 h at 37° C. in a 52μl reaction volume according to the protocol recommended by the supplierof the enzyme. The Omniscript reverse transcriptase was inactivated byheating at 93° C. for 5 min.

B. Q-PCR.

Quantitative PCR reactions were carried out in a 20 μl reaction mixture,containing 1X TaqMan™ Universal PCR Mastermix (PE Applied Biosystems cat#4304437), 0.12 mg BSA/ml, 900 nM of IGS4 specific forward and reverseprimers (IP14,963 and IP14,964), 250 nM of the IGS4 specific TaqMan™probe (IP14,962) and either 1.6 μl (IGS4) or 0.16 μl (GAPDH:glyceraldehyde-3 phosphate dehydrogenase) of the cDNA synthesis reactionas template. To set up the IGS4 standard curve a dilution series(10⁷-10¹ copies/reaction) of IGS4 plasmid ICCG 4320 was used whereas forthe GAPDH standard curve a dilution series of the human brain cDNAsynthesis reaction (0.16 μl, 0.016 and 0.0016 μl) was used as template.The 1X TaqMan™ Universal PCR Mastermix contained AmpliTaq Gold™ DNApolymerase, AmpErase™ UNG (uracil-N-glycosylase), dNTPs with dUTP,passive reference and optimized buffer components. IGS4 specific primersand TaqMan probe were designed using the Primer Express™ software (PEApplied Biosystems). Quantitative PCR reactions for human GAPDH werecarried out under identical conditions as described for IGS4 except thatGAPDH specific primers and TaqMan™ probe were used from the TaqMan™GAPDH control reagents kit (PE Applied Biosystems cat n° 402869;sequence information not available from PE Applied Biosystems).

PCR reactions were carried out in glass capillary cuvettes in theLightCycler™ instrument. After an initial incubation at 50° C. for 2 minto allow the AmpErase™ UNG reaction to proceed and activation of theAmpliTaq Gold DNA polymerase (95° C. for 10 min), reaction mixtures weresubjected to 40 cycles of denaturation (15 sec at 95° C.) andannealing/extension (1 min at either 60° C. [GAPDH] or at 68° C.[IGS4]). Quantification of experimental samples was carried out usingthe LightCycler Software version 3.0. A good linear relationship wasobtained between the amount of IGS4 plasmid and the release of reporterdye within the range of 10-10⁷ IGS4 plasmid copies. We also obtained alinear standard curve with the GAPDH TaqMan™ probe using the seriallydiluted brain cDNA. Relative GAPDH expression levels were in the rangeof 0.4 to 10.2% of that observed in skeletal muscle, which of alltissues tested had the highest GAPDH expression level. Relative IGS4expression levels were expressed as a proportion of the level detectedin spinal cord, which had the highest IGS4 expression of all tissuestested (FIG. 8). We also plotted relative IGS4 expression levels afternormalization for expression of the GAPDH house keeping gene (FIG. 8).

Example 5b Results

Q-PCR using an IGS4 specific TaqMan probe showed that highest expressionlevels (without normalization for GAPDH) were found in spinal cord. IGS4expression levels in spinal cord amounted to 11,467 copies mRNA/ng pARNA (assuming 100% efficiency of the cDNA synthesis reaction andassuming that pA RNA constitutes 2% of total RNA). High IGS4 expressionlevels were also found in brain (41% of spinal cord levels), skeletalmuscle (37%), cerebellum (31%), testis (19%) and in lung (12%) and heart(11%). Lower levels were found in fetal brain (5%), trachea (4%),prostate (2%) and thyroid (1.4%). After normalization for GAPDHexpression, the relative IGS4 expression pattern remained largelyunchanged with the exception of skeletal muscle, where the relativeexpression level dropped to 2% of that in spinal cord. As it is notclear whether normalization for GAPDH is a valid procedure (GAPDHexpression levels can be expected to vary more or less in differentcell/tissue types) we prefer to focus on the non-normalized relativeexpression levels.

These Q-PCR data seem to be in line with expression data from RNA array(Example 3) and Northern blot (Example 4) hybridization experiments inthe sense that testis, spinal cord and brain appear to be among the mostprominent expression sites. However Q-PCR analysis additionally showsimportant expression in a number of other tissues, such as skeletalmuscle, cerebellum, lung and heart.

The ligand neuromedin U has been proposed to be a neuropeptide orneuromodulator, without the knowledge of the specific receptor (DominJ., Ghatei M. A., Chohan P. and Bloom S. R. (1987), Peptides 8:779-784). Our investigation shows, that IGS4 is a novel member of theneuromedin U-receptor family being expressed in CNS and PNS regions, thegastrointestinal, immunological, genitourinary and cardiovascularsystem, skeletal muscle, thyroid, and lung.

TABLE 8 Overview of the PCR oligonucleotide primers and TaqMan probeused in the IGS4 Q-PCR reactions. SEQ ID NO: 32 IP 14,9635′-CCTCTTCAGCCTGGCGGTCTCTG- 3′ SEQ ID NO: 33 IP 14,9645′-GGAGGCGAAGCACACGGTCTCA- 3′ SEQ ID NO: 34 IP 14,9625′(FAM)-AGATGTGGCGCAACTACCC TTTCTTGTTCGGGCC-(TAMRA)3′

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of the relative positions of thedifferent cDNA clones that were isolated to generate the consensus IGS4cDNA sequence. 5′ and 3′ RACE primers that were used are also indicated(IGS4R# and IGS4F# respectively) as well as the position of ESTaccession n° N45474. Primer IGS4R6 was located within intron 1. Someclones (e.g. HNT2311, HNT2312 and HNT2253) were only partially sequenced(only the part that was sequenced is indicated). CONSENSUS A andCONSENSUS B denote the consensus sequence of IGS4 allelic types A and Brespectively. The nucleotide that was identified at each of the 4polymorphic positions is indicated (shaded boxes) for each clone. “S”indicates a sequence ambiguity in clones HNT2211 and HNT2212 and meanseither “C” or “T”. The coding area of IGS4A and IGS4B consensussequences is indicated with “**”. As there were some remaining sequenceambiguities in the 5′ end of the consensus sequence, the IGS4ADNA andIGS4BDNA sequences have only been taken from position 86 until the end

FIG. 2 Schematic representation of the relative positions of differentDNA database entries compared to the IGS4 cDNA sequence. The IGS4 cDNAsequence is indicated with the boxes (the position of the IGS4 codingsequences is indicated with the filled boxes). The relative position ofIGS4 exons 1-4 is indicated above the IGS4 cDNA sequence (“==”). Theparts of the genomic sequence AC008571 that encode exons 1->4 areindicated with AC008571a->d respectively. The position of thesefragments within the AC008571 sequence are: AC008571 a (13129-13908 ofthe reverse complement of AC008571), AC008571b (51676-51760 ofAC008571), AC008571c (79978-80103 of the reverse complement of AC008571)and AC008571d (83060-83728 of the reverse complement of AC008571).G05725 and G20615 are STS (sequence tagged sequence) entries whereasF05107, F05108, F07531, R13353, R13890, H11359, N45474, W61169,AI432384, W61131, AI023570, F01358, F03770, Z38158, R40869, R37725,H11333 are EST entries. The parts of genomic clones AQ019411 andAQ015065 that contain IGS4 exon 2 are indicated with “:”. The 5′ part ofEST sequences F05107, F05108, F07531, R13353, R13890 and H11359 which istotally different from the IGS4 cDNA sequence is indicated with “*”.AQ078563 is a genomic clone.

FIG. 3: IGS4 receptor activation by different Neuromedin U isoforms.CHOGα16-IGS4B cells were cultured in 96-well plates overnight and loadedwith Fluo-4AM. The receptor mediated Ca²⁺ changes were measured withFLIPR (Molecular Devices). Maxima of the fluorescence change detected bythe CCD camera were normalised to 1 and are depicted as counts.

FIG. 3 a: results for neuromedin U-8;

FIG. 3 b: results for neuromedin U-23;

FIG. 3 c: results for neuromedin U-25.

FIG. 4 Neuromedin U-23 induced intracellular Ca²⁺ mobilization inCHOGα16-cells expressing IGS4B. Application of 10 nM Neuromedin U-23 tothe cell lines CHOGα16-IGS4, CHOGα16 and CHOGα16 transfected with another orphan GPCR. Cells were cultured in 96-well plates overnight andlocated with Fluo-4AM. Receptor mediated intracellular Ca²⁺ changes weremeasured with FLIPR (Molecular Devices), depicted in counts detected bythe CCD camera.

FIG. 5 Human multiple tissue expression array using a human IGS4 probe.

FIG. 6 Northern blot analysis using an IGS4 probe.

FIG. 7 IGS4 expression analysis (MTE blot).

FIG. 8 Relative expression levels of IGS4 mRNA as compared to theexpression observed in spinal cord. Both non-normalized andGAPDH-normalized expression levels are shown.

1. A method of determining whether a substance is a potential ligand ofa neuromedin receptor comprising: (a) contacting cells expressing thereceptor chosen from SEQ ID NO:2, SEQ ID NO:4 SEQ ID NO:6 or SEQ IDNO:8, or the polypeptide encoded by a DNA insert contained in thedeposit no. CBS 102221 or the deposit no. CBS 102222 at theCentraalbureau voor Schimmelcultures at Baarn the Netherlands withlabeled neuromedin U in the presence and in the absence of thesubstance; or contacting a receptor membrane preparation comprising oneof said receptors chosen from SEQ ID NO:2, SEQ ID NO:4 SEQ ID NO:6 orSEQ ID NO:8 or a polypeptide encoded by the DNA insert contained in thedeposit no. CBS 102221 or the deposit no. CBS 102222 at theCentraalbureau voor Schimmelcultures at Baarn the Netherlands withlabeled neuromedin U in the presence and in the absence of thesubstance; and (b) measuring the binding of neuromedin U to thereceptor.
 2. A method of determining whether a substance is a potentialligand of a neuromedin receptor comprising: (a) contacting a cellexpressing the receptor chosen from SEQ ID NO:2, SEQ ID NO:4 SEQ ID NO:6or SEQ ID NO:8, or the polypeptide encoded by a DNA insert contained inthe deposit no. CBS 102221 or the deposit no. CBS 102222 at theCentraalbureau voor Schimmelcultures at Baarn the Netherlands withneuromedin U in the presence and in the absence of the substance; and(b) determining whether intracellular calcium levels are modulated inthe cell, wherein modulation of intracellular calcium levels indicatesthat the substance is a ligand of the receptor.